Featured Characters:

• Spider-Man (Peter Parker) (First appearance)

Supporting Characters:

• Dr. Curt Conners (Mentioned)
• S.H.I.E.L.D. (First appearance)

Antagonists:

• Electro (Max Dillon) (First appearance)
• Green Goblin (Norman Osborn) (First appearance)
• Doctor Octopus (Otto Octavius) (First appearance)
• R.H.I.N.O. (Alex O'Hirn) (First appearance)
• Sandman (Flint Marko) (First appearance)
• Venom (Eddie Brock) (First appearance)
  • Venom Symbiote (First appearance)

Other Characters:

• Street Thugs (First appearance)
• Goblins (First appearance)
• Unnamed Woman (First appearance)
• Roxxon Corporation (First appearance)
• NYPD Officers (First appearance)

Locations:

• Earth-TRN125 (First appearance)
  • Earth (First appearance)
    • United States of America (First appearance)
      • New York (First appearance)
        • New York City (First appearance)
          • Manhattan (First appearance)

Races:

• Humans (First appearance)
• Symbiotes (First appearance)

Items:

• Spider-Man's Suit (First appearance)
• Web-Shooters (First appearance)
• OZ Formula (First appearance)

Spider-Man is first seen fighting off a group of thugs attacking a woman. The woman calls him a freak and runs off. The Triskelion is seen in flames, caused by an explosion, which unleashed several symbiotes mixed with the OZ Formula. Spider-Man faces Venom and an army of symbiotes, and is then informed by SHIELD that Norman Osborn has captured several people. After stopping Norman, Dr. Conners creates a formula to destroy any symbiotes left in the city. In the end, Spider-Man is approached by the woman, who says he is not a freak.

• This game doesn't place in the Ultimate Universe continuity, despite having the Ultimate title and the art style.