If Big Band wins a fight, he has a chance of saying "I have to go now. My planet needs me." This is a reference to The Simpsons episode "The Itchy & Scratchy & Poochie Show".

One of Leviathan's possible quotes during Squigly's Daisy Pusher attack is "For I beheld Satan as he fell from Heaven like lightning!" This is a reference to one of Raul Julia's lines as M. Bison from the 1994 live action Street Fighter adaptation. This line was also originally used in Luke chapter 10, verse 18 in the Holy Bible.

During the 2013 Skullgirls Evo Breast Cancer Donation Drive, anybody who donated $50 USD could request a personal voice message from the voice actors of the game. One of the requests was for Peacock's voice actress, Sarah Anne Williams, to sing the song "Yakko's World" from the popular 90's cartoon Animaniacs.

Eliza is based and named after 2 people: Elizabeth Bathory, a Hungarian Countess and serial killer who believed that by using her victim's blood she would remain beautiful, mirrored by Eliza's own use of blood, and Elizabeth Taylor, the actress who played the title character in the 1963 film, Cleopatra.

When Filia beats Robo Fortune she will say "does she dream of electric mice?", a reference to the science fiction novel "Do Androids Dream Of Electric Sheep?" By Phillip K. Dick.

During the indiegogo campaign for Skullgirls Encore a reward was given to anyone who donated 1,000 dollars, the reward was to submit an NPC character to be included in the game.
Animator "Zone", who created Zone-tan and also worked on the game, was one of the "Patron Saint" backers. Zone-tan later appeared in the Glass Canopy stage.

The stage "Little Innsmouth" is a reference to the H.P. Lovecraft novella, "The Shadow Over Innsmouth".

If Valentine loses by timeout, she explodes into a mass of bones as if hit by a Mortal Kombat 3 fatality,with multiple skulls and ribcages.

Finishing the match with any Blockbuster attack will cause the background to change and display a portrait of the defeated character with a pained expression on their face. This is a reference to the Capcom fighting game JoJo's Bizarre Adventure, which features similar portraits for when a match is finished with a Super. The shaders that Skullgirls uses for its portraits in-game are also internally labeled as "JojosDeathPortrait" in the game's files.

On April 23, 2015, designer Earl "earlfriend" Gertwagen made a statement on the official Skullheart forums explaining that the reason why the game isn't available on either the Nintendo 3DS or the Wii U is because Skullgirls cannot run on the former and the lack of a publisher for the latter.

Parasoul's original wakeup animation (featured in the original 2012 Xbox Live Arcade/PlayStation Network release) showed the character's buttocks and underwear as she stood up from the ground. This was changed in the 2013 PC release, with her dress being longer and thus removing the partial nudity.

According to former lead design director Mike Zaimont, "Her skirt was actually the wrong length in the original version, it was fixed because it was off-model."

During Fukua's reveal trailer, the character Brain Drain is deciding which of the candidates featured in the video will become DLC and among those candidates is a dog. The dog gets rejected for being too cute and Brain Drain says "come back when you learn how to manipulate sand or something".

This is a reference to Iggy, a character from the JoJo's Bizarre Adventure series. In JoJo, Iggy is notable for being a dog who has the ability to manipulate sand.

Eliza's Story Mode text data commentary contains an ASCII picture based on fan art of Maya Amano from Persona 2. When questioned about it, programmer Jonathan Kim explained that many messages and pictures like that found in the game's code were the result of him venting off steam while working on the game, and that after the game had been datamined by fans, he was forced to remove the majority of those messages and pictures, with some exceptions like the Maya Amano fan art.

The game contains a character select portrait for Marie, the games final boss, suggesting that there were plans for her to be a playable character. She was later included as a candidate in a DLC voting campaign during a 2013 Indiegogo fundraiser, but lost to Eliza and Beowulf.

The "Politician Double" voice pack has a few specific pop culture references, including:
•The popular internet meme, "MURICA!"
•"WE'LL DO IT LIVE!", from Bill O'Reilly's infamous breakdown.
•"GREAT SKULLGIRL OR GREATEST SKULLGIRL?" is a reference to Steven Colbert, who jokingly asked if George W Bush was a "Great President or Greatest President?"
•"YOUR LIFE WAS FAIR AND BALANCED!" is a reference to the infamous Fox News slogan.
•"I DID NOT HAVE PUGILISTIC RELATIONS WITH THAT WOMAN!" is a reference to Bill Clinton's response after being accused of having an affair.
•"FOOL ME TWICE...uh...um...WON'T GET FOOLED AGAIN!" is a reference to Bush Jr. failing to remember the saying in a similar fashion.
•"HELP CONTROL THE PET POPULATION! HAVE YOUR PETS SPAYED OR NEUTERED!" is a reference to The Price is Right, when the host would say this exact line.

In Fukua's story mode, the boss fight with Filia will always be set to Nightmare difficulty no matter what difficulty is chosen.

The Diamond Deflector, a special move that Cerebella can do, is the only attack in the game that can reflect projectiles.

In River King Casino, it's possible to see Yuzuriha from Under Night: In-Birth, Matt and Woolie from the web series "Two Best Friends Play", and two dogs dressed as Chun-Li and Guile from the Street Fighter series.

One of Beowulf's blockbusters involves performing three uppercuts that each create an image of a howling wolf, which launches the opponent in mid air. After that, an image of a moon appears as he performs a body slam on the opponent. This is a reference to the infamous Three Wolf Moon T-shirt on Amazon.com.

The stage Gehenna is named after a place outside ancient Jerusalem where the worshippers of Baal had child sacrifices and was described as "filled with the blood of innocents".