Cuphead's appearance at E3 2014 features an unused boss referred to by fans as the "Demon Bat". It is unknown why this boss went unused, but it's most likely that the developers decided that certain phases from the Demon Bat's fight may have fit better with another boss in the game, as this design philosophy was mentioned in an IGN interview. This is evidenced by Djimmi the Great's third phase in the final game, where he transforms into a sarcophagus and spawns minions to go straight for the player, being nearly identical to one of the Demon Bat's attacks in a pre-release screenshot from E3 2014. Another underlying reason the boss may have been scrapped is due to its pink lips and black skin unintentionally resembling offensive blackface characters from the cartoons Cuphead was inspired by.

In The Delicious Last Course DLC, when transitioning to his second phase, Chef Saltbaker uses a red mushroom to grow into a giant, a clear nod to the Mario franchise. Despite the mushroom’s reddish-pink hue, it doesn’t count as a pink object that can be parried off of.

In "The Devil & Ms. Chalice", the final episode of the animated series "The Cuphead Show!", the last scene of the episode features the Devil's Casino in its only appearance in the show. The final shot of the series shows Cuphead, Mugman and Ms. Chalice running towards the casino, referencing the opening of the game. While not confirmed, this also implies that the show is, at least in broad strokes, a prequel to the game.

In "The Delicious Last Course" DLC, there is a puzzle on the map for Isle IV involving a trio of contest winners towards the back of the Isle and a set of 9 gravestones towards the front of the Isle. Completing this puzzle will unlock Spoiler:a secret boss fight that takes place in a dream world held up by the skeletal remains of the Devil (seen in claymation), where you fight angel and demon variants of the Devil on both sides of the stage. However, they each switch places back-and-forth every time you turn left or right, presumably due to Cuphead's interactions with the Devil in the main game preventing him from seeing an Angel. This boss fight was originally designed as a second phase for the Devil's fight in the main game, but ultimately went unused until the release of The Delicious Last Course. A primitive version of the phase can be found in the game’s data. This boss cannot be refought after beating it. Beating it will unlock the Cursed Relic as a charm as well as the achievement "A Horrible Night to Have a Curse", a reference to the text box seen during the day-to-night transition from Castlevania II: Simon's Quest.

Version 1.2 of the game added secret alternate phases to three boss fights, two of which being ideas that were originally scrapped before the game's release:
•Botanic Panic!: Spoiler:By not attacking Ollie Bulb the onion, he will retreat back into the ground and skip to Chauncey Chantenay the carrot's phase featuring an additional mini-boss, a spinning radish named Horace Radiche who despite being completed before the game was released, originally went unused.
•Pyramid Peril: Spoiler:If the player shrinks into the mini-plane form just before the fourth phase of the fight, Djimmi the Great will scan the player and pull up a marionette featuring a dinosaur, a miniature version of Dr. Kahl's Robot and a stone statue, before tugging a longer string and pulling up a smaller version of Cuppet. This skips the fourth phase and goes directly into a harder version of the final phase where the smaller Cuppet will act as a turret firing at the player counter-clockwise to the spinning Eye of Providence pyramids.
•Dramatic Fanfare: Spoiler:By parrying off of the pink star projectiles, the player can land onto two hanging cherub props that control the chandelier in the background of the first phase. Standing on one will gradually lower it until a dinging sound is heard and the chandelier becomes slanted. Doing the same to the other cherub will cause the first phase to abruptly end and trigger an alternate storyline to the fight that was originally unfinished by the time the game was released. After both cherubs are lowered, Sally Stageplay's newlywed husband is crushed by the chandelier. Instead of changing the scenery to Sally's home, the second phase of the fight takes place at St. Timothy's Nunnery, implying that Sally became a nun. In place of Sally's husband worrying in the bushes for her is the Priest that oversaw their marriage, and a Nun takes the place of Sally's children to throw rulers at the player. In the third phase, a god form of Sally's husband accompanies Sally's goddess form and attacks by summoning a cherub, portrayed by Sally's son from the normal second phase of the fight, to push a spinning cardboard fireball towards the player. The fourth phase of the fight is unchanged from the normal path, but with the Nun appearing in the background in place of Sally's husband and children when on Expert mode.

One boss fight in "The Delicious Last Course" DLC also features a secret phase:

•Doggone Dogfight: Spoiler:During the second phase with the circling jetpack puppies, their jetpacks will produce white exhaust fumes when they have plenty of health, with the more damage taken turning it into gray exhaust to indicate that they have low health. If all four jetpacks are spewing gray exhaust at the same time with none of them having been defeated, the Dog Chinook will appear and eat them before grabbing the plane as if it were the normal third phase. However, the puppies and the Chinook pilot will appear in portholes on its paw pads instead of the laser satellites, where they throw regular and pink Pineapple Mint grenades that explode into shrapnel when they are hit. The Chinook pilot also appears and calls for the vessel to shoot homing fire hydrant missiles.

According to animator and designer Tina Nawrocki, Baroness Von Bon Bon's design was inspired by Bebe Daniels, Betty Grable, Loretta Young, and Joan Crawford (specifically her appearance in the 1932 film "Letty Lynton"); actresses who were around during the early 20th century, as well as Marie Antoinette, the last queen of France before the French Revolution, whose execution by guillotine inspired the fourth phase of her boss fight where she throws her own head as a projectile towards Cuphead.

If the game detects that the game is pirated, a pitch-shifted and reversed version of the main theme song will play on the title screen. When this song is plugged into a spectrogram, the end of the song will form The Devil in the 3 forms of his boss fight - Regular, skeletal and giant face.

Found within the game's files is an unused title theme for Cuphead with lyrics. This may have been composed during early stages of the game's development, as the lyrics describe a completely different plot from what was featured in the final game.

According to Jake Clark, when he was designing Cala Maria, The character went through a notable amount of changes before settling with the final design.

Before being settled for having a octopus on her head. Her face originally had fish fins on the side of her face, she had various different types of crowns (along with different hair styles) a starfish was considered to be used at one point, she had a round earring on her nose, a crab tattoo on her right arm, and wearing gloves.

It was also considered for her to hold a weapon(A sword and an anchor). As well the following for her attacks. Cala using her tail, summoning swordfish, a giant crab, and using a title wave.

In the games files, you can find an unused boss based off of a Pachinko machine that goes by the name "Pachi-Pachi".

The developers planned to surpass the Guinness World Record for number of boss battles in a run and gun game by having over 30 to the record's 25.

According to animator Jake Clark (who animated most of the bosses in the game), the mermaid boss Cala Maria was inspired by not only Betty Boop, but also Disney's Ariel (for the bra) from The Little Mermaid, and a female moth character from the Disney Silly Symphony cartoon "Moth and the Flame".

In the background of Botanic Panic!, there's a bag that says 'Acme Grow'. This is a reference to the heavy use of the word "Acme" (an Ancient Greek word meaning "the peak", "zenith" or "prime") as a generic corporate name in media from the silent film era onwards, most prominently in Warner Bros.' "Road Runner and Wile E. Coyote" Looney Tunes shorts.

In 2019, Studio MDHR and the multinational automotive company Tesla announced that a special port of Cuphead would be released for the Tesla Arcade digital store and would be playable on the company's Model S, Model X, and Model 3 vehicles. Due to storage limitations with the cars, the Tesla Arcade version only includes the stages in Inkwell Isle I. The game can only be played with a controller plugged into each vehicle's USB port, but can be played cooperatively with two players controlling Cuphead and Mugman. As part of the announcement, Studio MDHR released commemorative artwork of Cuphead and Mugman standing with a Model 3 car.

On August 24, 2017, a video was uploaded by gaming news outlet VentureBeat where lead writer Dean Takahashi recorded a gameplay demonstration of him playing the Gamescom 2017 demo for Cuphead due to him being the only one on staff at Gamescom. He specializes in general industry articles, strategy games and first/third-person shooters, and normally does not cover platformers or sidescrolling action games, because by the outlet's own admission he was extremely bad at them. This footage became notorious for the first two and a half minutes where he struggles to complete the game's tutorial, before struggling to play for another 23 minutes under conditions that were made intentionally easier for the game's demo such as increased health and instant access to some stronger unlockable charms like Spread. VentureBeat knew the footage was bad, but uploaded it anyways and drew attention to Takahashi's poor gameplay in the video title, calling it "shameful". However, VentureBeat initially did not explain the full context of the footage in the video description, and due to Gamescom being held one month prior to Cuphead's release, the clip was passed around out of context leading people to believe he was doing a full review of the game and trying to make a point of it being too difficult. In reality, the video was posted alongside an article about the demo by Takahashi to VentureBeat that regularly acknowledges his poor skill at the game; he also called Cuphead a fun game that showed "why making hard games that depend on skill is like a lost art". Regardless, the footage still drew extreme negative backlash and harassment towards him and claims that he was unfit to be a game journalist. Takahashi's response to the controversy spurred more controversy after he accused people attacking the footage of being connected to the 2014 #Gamergate movement, when one week prior to responding, he published an article promoting the idea of a "leisure economy" that stems from game journalists among others being paid to play games, and promoting the fact that he had been reviewing games for 21 years up to that point.

According to a GameSpot interview with the game's lead designer Jared Moldenhauer, he confirmed that the presence of a plush lizard and a plush wolf alongside Mr. Chimes the monkey toy boss is an homage to George the gorilla, Lizzie the lizard, and Ralph the wolf from the arcade game Rampage.

One of Cagney Carnation's attacks in his final phase has him spitting dandelion seeds. As of version 1.1.3, when a player gets hit by one of these seeds, the screen blurs and the colors shift briefly. On Studio MDHR’s official website page regarding the update, the final bullet point says “Touch fuzzy, get a little dizzy”. This attack is a reference to Level 1-7 “Touch Fuzzy, Get Dizzy” from Super Mario World 2: Yoshi's Island.

In the The Delicious Last Course DLC, if the player loses to Chef Saltbaker on his 4th phase his taunting quote will be "The mark of my salinity shall scar thy fired glaze!". This is a reference to Street Fighter III: 3rd Strike, where the game's final boss Gill has a pre-battle dialogue that says "The mark of my dignity shall scar thy DNA".

After the release of The Delicious Last Course DLC, an Easter egg was added where after spinning in place three times, Djimmi the Great will appear and say "You've freed Game Djimmi! I grant thee infinite wishes on Simple and Regular Modes!" This quote references the Game Genie peripheral, and his wishes grant the player double the HP for the next boss fight.

The Latin American Spanish translation of The Delicious Last Course DLC changes The Pawns mini-boss death quote to "Pasito a pasito, suave suavecito, me llevo la victoria, poquito a poquito". This is a reference to a line from the song "Despacito" by Luis Fonsi and Daddy Yankee: "Pasito a pasito, suave suavecito, nos vamos pegando poquito a poquito."