Mario Tennis on the Game Boy Color and Mario Kart Tour are the only Mario multiplayer games where Mario is unlockable. In terms of single-player games, Mario is also unlockable in the single-player mode of Mario Golf on Nintendo 64, as well as Super Mario 64 DS.

During the height of Super Mario 64 DS' popularity, a popular rumor was that Waluigi was hidden as an unlockable character - this rumor was inspired both by the presence of Wario and Luigi without their combined counterpart, and a set of coincidences within the game and its supplementary material:

• The character switching room having 4 doors, one for Mario; one for Luigi; and one for Wario, along with a final, empty room (this room actually contained a Power Star).
• 4 bunnies existing in the game: a yellow bunny who appears when playing as Yoshi, a pink bunny who appears when playing as Mario, a green bunny who appears when playing as Luigi, and an orange bunny who appears when playing as Wario. The manual's page on the mini-games that bunnies are used to unlock shows a purple bunny that doesn't appear in the game.
• The title screen and floor of the Rec Room having a colour scheme based on yellow (Wario's color), green (Luigi and Yoshi's color), red (Mario's color), and purple (Waluigi's color).
• The image of Peach's Castle on the file select screen having what appears to be an open window that does not exist in the game, which many assume is where Waluigi would be hiding.

The popularity of these theories would birth an infamous hoax known as "Purple Prizes", a graphic created by Andrew Brown of Nintendo World Report in an attempt to rebirth the mystery he felt as a child from trying to find Luigi in the original Super Mario 64 for the younger Nintendo DS generation, and posted it to DeviantArt and a set of gaming forums under the false pretense of being a scan from an unnamed magazine. Although he clarified that the picture was fake a mere 2 days later, the image still spread online like wildfire, and was misreported as originating from Nintendo Power or IGN. Brown noted that some attempts to debunk flaws in the image were not based on the actual and plentiful flaws he acknowledged the image as having, but rather misconceptions such as Waluigi's yellow gamma being a C or accusing certain images of being based on the original Nintendo 64 game instead of the DS remake.

According to former Nintendo employee Motoi Okamoto, around the time of this game's development, Shigeru Miyamoto would begin 6-hour-long nighttime crunch sessions during the development of Super Mario 64 DS by exclaiming "It's Mario time.":

In the only instance of a character having a move exclusive to VS Mode, when Yoshi is wearing Wario's Cap, he can perform an additional move called the "Swing and Fling" by picking up a stunned Yoshi, spinning around in a circle and throwing him. This move is only mentioned in the game's manual. Wario's animations when using this move share similarities to the "Wild Swing-Ding" move featured in Wario World, although the Swing and Fling does not take over spinning automatically.

In the European and American versions of the game, King Boo has an empty text box displayed when he is confronted by Yoshi in Big Boo's Haunt. This means Yoshi could have originally entered Big Boo's Haunt without the requirement of selecting Mario, Luigi, or Wario.

The Korean version of the game lacks a backlight setting and the Rec Room seen in other versions, possibly due to the gambling minigames which South Korea doesn't allow in video games.

In the South Korean version of the game, there's a Yellow ! Block that appears on the castle roof right next to the Red ? Block. Hitting it from underneath will give the player an infinite amount of extra lives and will not disappear, allowing them to easily acquire 100 lives. This was removed in all other versions of the game.

Found within the game's data is an unused Red Koopa Troopa that can only appear through hacking methods. Unlike the green Koopa Troopas found in the game, the red ones will attempt to run at the player and knock them around similar to Bullies. When knocked out of their shells, they'll slowly walk back into them instead of panicking like the green ones do. In addition, their shells cannot be ridden. Instead, they will only slide along the ground like in previous Mario games, killing enemies in their path until they break against a wall. If a Red Koopa Troopa is eaten by Yoshi, he'll spew out fire like he does when eating one in Super Mario World.

By alternating between Mario and Yoshi's drawing icon three times on the title screen, Mario will transform into a drawing of Luigi.

Coincentration's sprite set contains some Super Mario Advance 4 sprites, which aren't used in the final game. Coincentration's internal name is "wrecking_crew", a reference to the NES game of the same name. The mini-game uses this name in New Super Mario Bros. as well.

In the mission "Blast to the Stone Pillar" in Jolly Roger Bay, the Power Star is contained within a box in the Western versions of the original game, but not in the Japanese version. This was likely changed during localization to make the star's position less obvious. However, all versions of the Nintendo DS remake leave the Power Star out in the open.

The Nintendo 64 version of the room containing the entrance to The Princess's Secret Slide is still part of the in-game's castle model, complete with a sign and a functional warp to the Secret Slide. The door leading to this room was simply changed to lead to the new version of the room.

"Wing Mario Over the Rainbow" is the only course that does not have a Luigi Cap, even when Luigi is unlocked.

In the game's data, replacing a file called "0000_PLAYER" with another file spawns a clone of the player that follows his movements. It takes on the appearance of Mario, but will shift into a different character when hit (spawning with a Mario cap).

The Bob-Omb Battlefield mission "Shoot To The Island In The Sky" from the Nintendo 64 version was removed in Super Mario 64 DS, and replaced with the mission "5 Silver Stars".

At the start of the game, Yoshi can be seen sleeping on top of the castle. This is a reference to where you found him after you got all the stars in the original Super Mario 64.

When first announced, Super Mario 64 DS was named "Super Mario 64 x 4", and seemed to feature gameplay where the player would utilize all four characters at the same time. This early build was shown at E3 2004 before being altered to be more like the original.

In the minigame "Loves Me/Loves Me Not", the tune that plays during a "REALLY LOVES ME!" is the Recorder theme from The Legend of Zelda.

Each boss in the game has a specific piece of dialogue based on who you're fighting them as. Even though Yoshi can't actually fight Chief Chilly, there is still dialogue programmed in for that fight.

In the Japanese version of the original game, the painting for Jolly Roger Bay originally had a blue frame with bubbles. This was changed to a pirate ship with a gold frame in the international release of the original game. In the Nintendo DS remake, the painting reverted back to the original blue-framed form.