- Beetle (First appearance)
- Jack O'Lantern (First appearance)
- Alistair Smythe (First appearance)
- Scream (First appearance)
- Doctor Octopus
- Tinkerer (First appearance)
- Green Goblin
- Fantastic Four (First appearance)
- The manual notes that the Fantastic Four's Human Torch was named after an "android hero of the 1940's," referring to Jim Hammond, the original Human Torch.
- Although based on Spider-Man: The Animated Series, the game included many characters who never appeared on the show, such as Beetle, Scream, and Jack O'Lantern. Additionally, the show's version of Norman Osborn wouldn't become the Green Goblin until the year after the game's release  and Spider-Man wouldn't meet the Fantastic Four until the Secret Wars. If the game takes place on Earth-92131, it would have to take place long after the Animated Series' ending.
- The Web-Armor power-up may be a reference to the Spider-Armor MK I.