In the game's files are unused animations for Mario that include:

• Mario putting on his hat faster.
• An alternate animation for when Mario rides a Koopa Troopa shell.
• Mario changing from a free fall to a forward flip.
• Either Mario crying or reaching for something in his pockets.
• Mario's unused running animation
• Mario motionless while sliding
• A forward sideflip.

The game's producer and director Shigeru Miyamoto was asked if he had any references, or anything else he relied on when making all of Mario’s various animations. He responded:

"We tried out a lot of different things using motion capture, but ultimately we ended up doing it all by hand. We created a “skeleton” for Mario that was the basis of his movement."

The skybox of Wet-Dry World, originally believed to be a stock photograph of the hillside town of Casares, Spain, was later found to be a composite of altered photographs of the town of Shibam, Yemen. The sole exception is the red building near the top of the picture, which was identified as the Great Mosque of Muhammad Ali Pasha in the city of Cairo, Egypt. The location fits the idea of Wet-Dry World, because Yemen is a desert country that, like other Middle Eastern countries, experiences sporadic large-scale flash floods.

In the European version, the demo clip depicting Mario collecting a ring of coins in Whomp's Fortress sees him grab one less coin than in all other versions of the game, resulting in two coins being left on-screen instead of just one. Oddly, this is the only change made to the game's demo videos between regions.

The game's smoke texture, labelled "gas_b5" in the source code, was intended to be much more detailed in appearance, but ended up corrupted in the final game due to being encoded in the RGBA16 format rather than the intended IA16. While the glitched texture would theoretically look like a swarm of multicolored pixels as a result of this error, it instead appears as a clump of black pixels due to the game displaying it without any colors, resulting in it not looking out of place. Modifying the game's code to convert the texture to the IA16 format results in it properly displaying.

By standing against the wall between the left and middle paintings for Cool, Cool Mountain and using the first-person view, you can clip the camera through the wall to find what appears to be a large red "F" floating in the void. This is actually part of the entrance to Bowser in the Dark World, a course on the same floor, seen from below.

Among the files included in the Gigaleak, a massive 2020 content leak of internal data from Nintendo, are assets associated with Super Mario 64 depicting a fully-coded enemy named Motos, which is absent from the final game. The enemy is programed to chase Mario, pick him up, and throw him similarly to Big Bob-omb and Chuckya. Like those characters, Motos can be picked up and thrown, but unlike them, it can only be defeated by being thrown into lava, similarly to the Bully and Big Bully. This weakness implies that Motos was originally intended to appear as an enemy in Lethal Lava Land and Bowser in the Fire Sea, which is corroborated by the fact that various other assets associated with the levels feature "motos" in their filenames.

Further analysis of the source code indicates that Motos were intended to be connected to Bullies, which are labeled "Otos" in the source code and use sound effects that are also associated with Motos. Additionally, the code contains a filename for an "icemotos" miniboss in Snowman's Land, and the "Bully the Bullies" star in Lethal Lava Land is internally named "bigmotos," implying that at least two variants of Motos were conceived as minibosses before being replaced with Big Bully and Chill Bully in the final game.

Additional data in the Gigaleak indicates that at some point in development, Motos was reconfigured into a boss version of the Chuckya enemy, labeled "Katsugikun" in the source code, before being scrapped altogether. Unlike Motos, no assets for Katsugikun exist other than a filename and behavior code.

Starting with Super Mario 64, the Koopa Troopa's voice is an incredibly short snippet of "HUMAN, YELL - YELL: FEMALE", a sound effect of a woman gleefully screaming from the Best Service Voice Spectral Volume 1 sound pack, modified into different pitches and speeds. In some games, a different snippet from the sound effect is used to represent Koopa Troopas screaming or being frustrated.

According to Shigeru Miyamoto, the design of the Deku Baba enemies in The Legend of Zelda series were a cross between a dog and the giant snapping Clams from Super Mario 64.

A 2017 study conducted by a team of Canadian neuropsychologists found that playing Super Mario 64 increased grey matter in the brains of older adults, potentially helping to combat dementia and other neurological disorders. According to the study:

The first-ever footage of Luigi as a playable character in Super Mario 64 was discovered through an old VHS tape in 2023. The footage was captured by a Japanese broadcaster recording footage of Nintendo's 1995 Space World trade show in Japan for a TV program covering the event. Despite this, the footage of Luigi is not actually the primary focus of the clip and as such can barely be seen at all. The video shows Luigi falling back down after performing a spin-jump in what appears to be a red and white test area.

The Idea for Metal Mario may have come from an interview with Shigeru Miyamoto published in 1991, in Nintendo Power's Mario Mania Player's Guide, Miyamoto reportedly said: "Who knows what Mario will look like in the future? Maybe he'll wear metallic clothes!"

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.

There are murals on the pillars in Bowser in the Sky depicting the original fight with Bowser from Super Mario Bros.

The penguin found during the 3rd star of the "Cool, Cool Mountain" level changes after 120 stars are found. After obtaining all the stars, the penguin is found much fatter than before from not doing races lately. He can be raced again, but there is no additional reward for beating him.

When you catch MIPS the bunny in the castle basement, he references Alice In Wonderland. Just like the White Rabbit, he says he's late. Then he says says he'll give you a star, but he has an important date to get to.

In early development of the game when Nintendo were working on character movement and camera angles, they only had two characters; MIPS the rabbit and Mario. MIPS was planned to be just a testing dummy, but made it into the final game because they didn't want to part with him.

MIPS, the rabbit in the castle basement, is named after the MIPS (Microprocessor without Interlocked Pipeline Stages) microprocessor. The Nintendo 64's CPU runs on a MIPS Microprocessor, so the name was chosen for the rabbit.

As early as January 1997, a cancelled sequel for Super Mario 64 was in development, commonly called "Super Mario 64 2" or "Super Mario 128" (not to be confused with the tech demo of the same name) for the failed Nintendo 64DD expansion. Only one demo was ever made, but was never shown to the public. Luigi was planned to feature in it as a playable character, and anywhere from two to four player co-operative play was planned. Shigeru Miyamoto once said he hoped to make the game as complex as Banjo-Kazooie, and joked that the box art for the game would be green if the demand was strong enough for Luigi.

If you jump up and down in a shallow pool of water, you can occasionally see a fish jump out of the water with you.