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- X-Men (First appearance)
- Beast (Henry McCoy) (First appearance)
- Dr. Watts (First appearance)
- Lizard (Curt Connors)
- Electro / Hyper-Electro (Max Dillon) (First appearance)
- Shocker (Herman Schultz) (First appearance)
- Sandman (William Baker) (First appearance)
- Beetle (Abner Jenkins) (First appearance)
- Hammerhead (First appearance)
- X-Men (First appearance)
- Captain America (Steve Rogers) (Mentioned)
- Elektra (Elektra Natchios) (Mentioned)
- Thor (Photo)
- Scorpion (MacDonald Gargan) (Cameo)
- Rhino (Aleksei Sytsevich) (Cameo)
- Mysterio (Quentin Beck) (Cameo)
- Doctor Octopus (Otto Octavius) (Cameo)
- Jade Syndicate (Cameo)
- Dr. Stroser (First appearance) (What If? mode)
- Hobgoblin (Roderick Kingsley) (Mentioned)
- Hulk (Bruce Banner) (Mentioned)
- Iron Man (Tony Stark) (Mentioned)
Races and Species:
- Bio-Nexus Device
- Spider-Man's Suit
- Spider-Armor MK I
- Dusk's Suit
- Prodigy's Suit
- Insulated Suit
- Alex Ross - Red
- Alex Ross - White
- Venom 2 - Earth X
- Negative Zone
- Symbiote Spider-Man
- Spider-Man's 2099 Suit
- Captain Universe Spider-Man
- Amazing Bag Man
- Scarlet Spidey
- Ben Reilly Spider-Man
- Quick Change Spidey
- Civilian Peter Parker
- Battle Damaged
- Mjolnir (Photo)
- Doctor Octopus' Tentacles
- Rhino Armor
- Scorpion's Suit
- Mysterio Suit
- Beetle Armor MK II
- Shocker's Vibro-Shock Gauntlets
Electro has been collecting parts for his device.
- Rino Romano - Spider-Man (Peter Parker) / Palooka / Police Announcer / Bank Thug
- Dee Bradley Baker - Electro (Hyper-Electro) / Computer 3 / Lizard / Hammerhead / Beast / Rhino
- Kathryn Fiore - Intro-Recap Announcer / Dr. Stroser / Computer Voice
- Daran Norris - Sandman / Beetle / Public Address / Shocker / Professor X / Scorpion / Mysterio
- Jennifer Hale - Rogue / Dr. Watts / Computer 2
- Stan Lee - Stan Lee
- One major difference of this game from its predecessor was the presence of ground levels. This game presented levels that are city streets confined within a grid.
- Although the gameplay is relatively similar to the first game, the basic punch and kick combo moves are changed, doing away with the two-handed uppercut / mule-kick for the third strike. Additionally, in this game Spider-Man was given the ability to fire a web-ball in mid-air.
- Training mode takes the player to the X-Men's Danger Room where Rogue and Professor X teach you what Spidey needs to know.
- Character models were refined with hand animations, whereas in the first game every character had their hands clenched into a fist at all times.
- During the cutscene after Spider-Man beats Shocker, he swings away to Hangar 18. Before he does that, he says, "I don't need pointy ears and a cape to tell that this is another heist." A reference to Batman. Spider-Man's voice actor, Rino Romano, wound up voicing Batman years later in the animated series The Batman.
- The cutscene where Hammerhead discusses the importance of teamwork in their plan to stop Spider-Man is quoted from Robert De Niro's character Al Capone in the movie The Untouchables. At the end, however, Hammerhead does not bludgeon a fellow mobster to death with his baseball bat, instead bashing a glass of red wine which spills over a newspaper photo of Spider-Man, which probably represents the blood which the mobster had spill.
- In the final cutscene where Hammerhead, Shocker and Electro are jailed, Hammerhead and Shocker play cards but end up frustrated since all they've played was poker. Shocker asks the prisoners from the front cell if they know how to play Go Fish, these inmates are Doctor Octopus, Scorpion, Rhino and Mysterio, still incarcerated after the events from the first videogame.
- The September 11 attacks happened during development, as a result, references that are similar to the attacks were removed and the World Trade Center was replaced with a skyscraper with a bridge on it.