The first boss in the game, the Stinger, has a simple way to be skipped entirely. To do this, the player must run up to the Stinger and drop an item in front of it, then turn around and wait for the boss to lunge at them. If the player picks up the item they just dropped at the right time, they’ll become invulnerable, and the scorpion’s attack will go straight through them. The player can then back out of the menu and simply run past the Stinger to safety.

When the players defeats 16 bats and the Infected Bat in the church, they will unlock 2 trophies/achievements titled "Winged Spawn of Satan" and "Die, Devil Bird!". These are a reference to Ace Ventura's quotes, when he enters the bat cave to look for Shikaka in the film Ace Ventura: When Nature Calls.

The idea of not having "item boxes" in the game was based on "Sweet Home". The team wanted to remove it, so that it would feel a lot harder to play.

In addition to Rebecca's western costume originally available in the game, every new costume added in the game's HD port is a reference to one of Capcom's previous games.

• S.T.A.R.S. 1996 (Rebecca): Rebecca's costume from Resident Evil
• S.T.A.R.S. 1997 (Rebecca): Resident Evil Director's Cut's Arrange Mode
• S.T.A.R.S. Prototype (Rebecca): The unreleased Nintendo 64 version of RE0
• Sportswear (Rebecca): Resident Evil 2, easter egg
• Cheerleader (Rebecca): Deadly Silence, alternate outfit
• Western (Rebecca): Resident Evil Remake, alternate outfit
• Nurse (Rebecca): Resident Evil: The Mercenaries 3D, alternate outfit
• Battlesuit (Rebecca): Version of Jill's outfit from Resident Evil 5
• Prisoner (Billy): Cody Travers from Final Fight
• Wolf Force (Billy): The Japanese cover art of Mercs

Additionally, three t-shirts are available for Rebecca. They are the winner designs by fans from Capcom's In-Game T-Shirt Contest. One of these was done by former SNK and Capcom's artist Shinkiro (real name Toshiaki Mori). Another one makes a reference to one of Barry's lines from the first Resident Evil.

While it was still being developed on the Nintendo 64, the game was going to have multiple endings depending on which character(s) were still alive at the end of the game. This meant both Rebecca and Billy could have gotten killed at some point in the game. The idea was scrapped due to the possibility of Rebecca's death contradicting the series' timeline.

During the development of Resident Evil Zero, the developers toyed with the idea of including running zombies in the game. They didn't make it into the game's final release, but the idea was reused as the Crimson Heads of the 2002 Resident Evil remake.

Early on in the Gamecube and Wii versions of the game, inside the train, there are illegible signs above the car doors. They were illegible in the original resolution but were removed from the HD release where they would have become clearly visible. The original sign simply said "Dining Car" but was reused for all the doors. There's also a blurred poster on the train, which is actually a picture of one of the game's programmers, this was also changed for the HD version for similar reasons.

Capcom accidentally revealed the game's HD rerelease months before its official announcement while showcasing the theme for Resident Evil HD Remaster for PlayStation 4. A game icon for the remaster could be seen in the menu.

Satoshi Nakai, who worked on Resident Evil Zero in its early stages of development, originally proposed civilians as the protagonists for the game. The two main characters would've been Annie and a significantly different looking Billy.

There is a pink herb found in the files of Resident Evil 0 that is not available in the game. Its usage is unknown and it does not appear in any of the other games unlike red, blue, yellow and green herbs.

In the N64 beta of the game, STARS Bravo Team member Edward Dewey is found missing a right hand, after being attacked by Cerberus dogs. This would explain the severed hand holding a gun that would be found by Joseph Frost in the original Resident Evil.

In the final game he still has both hands as the remake of Resident Evil no longer has Joseph finding a severed hand.

Resident Evil Zero was originally planned for the Nintendo 64 in the year 2000. Rebecca is shown to wear a beret similar to Jill Valentine from Resident Evil 1, and Billy's facial features were totally different from his final design.

Most of the characters and settings from the Gamecube version are in the N64 version, like the opening fight on a moving train. It was in development during the last years of the N64, so instead of finishing it, Capcom scrapped the build and developed it for the GameCube.

Billy Coen's tattoo says "Mother Love", a reference to the song of the same name by the band Queen.