- Mary Jane Watson-Parker
- Daily Bugle
- J. Jonah Jameson
- Robbie Robertson (Master System and Game Gear versions only)
- Kingpin (Wilson Fisk)
- Doctor Octopus (Otto Octavius)
- Electro (Max Dillon)
- Lizard (Curt Connors)
- Sandman (Flint Marko)
- Hobgoblin (Roderick Kingsley)
- Venom (Eddie Brock)
- Vulture (Adrian Toomes) (Sega CD version only)
- Mysterio (Quentin Beck) (Sega CD version only)
- Bullseye (Lester) (Sega CD version only)
- Typhoid Mary (Mary Walker) (Sega CD version only)
- Kingpin (Wilson Fisk) (Spider-Man & MJ killed alternate ending)
- Kingpin (Wilson Fisk) (MJ killed alternate ending)
- Doctor Strange (Stephen Strange) (Master System and Game Gear versions only)
- Spider-Man (Peter Parker) (Only appearance; dies) (Spider-Man & MJ killed alternate ending)
- Spider-Man (Peter Parker) (MJ killed alternate ending)
- Mary Jane Watson-Parker (Only appearance; dies) (Spider-Man & MJ killed alternate ending)
- Mary Jane Watson-Parker (Only appearance; dies) (MJ killed alternate ending)
Races and Species:
- Earth-813191 (Only appearance)
- Earth-831911 (Only appearance)
- Earth-931811 (Only appearance)
- Spider-Man's Suit
- Doctor Octopus' Tentacles
- Goblin Armor
- Pumpkin Bombs
- Vulture's Wings
- Mysterio Suit
- Disintegrator Cane
- Cloak of Levitation
The Kingpin has framed Spider-Man for the theft of a nuclear bomb, and plans to detonate it in a twenty-four-hour period, unless Spider-Man is able to find all the keys needed to disable it. Subsequently, Mary Jane is kidnapped by Venom.
- In the Mega-CD and Mega Drive games, there are alternate endings depending on if the player is able to rescue Mary Jane in time and or defeat the Kingpin.
- Although this is the first official Spider-Man videogame for Sega Consoles, this is not the first time he appeared in a Sega game. That honor goes to The Revenge of Shinobi released in 1990. Spider-Man appears as the first part of a two-phase boss battle in Stage 6, Chinatown. After he takes enough damage, Spider-Man leaves the fight and is replaced by Devilman from the manga of the same name. In the original Japanese version of the game as well as the version featured in the Sega Smash Pack, this boss battle pitted the player against a shapeshifting ninja named "Metamorpher", originally appearing in a form similar to Spider-Man before morphing into a form that resembles Batman. When Sega obtained the rights to Spider-Man for The Amazing Spider-Man vs. The Kingpin, the boss was altered to be Spider-Man and Devilman in all future releases, starting with the American release.
- In the Master System and Game Gear versions of the game, Doctor Strange assists Spider-Man during the cutscenes, telling him where the location of the villains are.
- In the Game Gear version of the game, one can follow a series of directions to equip Spider-Man with the symbiote costume.