Featured Characters:

• Spider-Man (Peter Parker) (Last appearance)
• Venom (Eddie Brock (Last appearance)

Antagonists:

• The Jury (First appearance)
  • Sentry (Curtis Elkins) (First appearance)
  • Ramshot (Samuel Caulkin) (First appearance)
  • Screech (Maxwell Taylor) (First appearance)
  • Bomblast (First appearance)
  • Firearm (First appearance)
  • Wysper (Jennifer Stewart} (First appearance)
• Life Foundation Symbiotes (First appearance)
  • Riot (Trevor Cole) (First appearance)
  • Phage (Carl Mach) (First appearance)
  • Lasher (Ramon Hernandez) (First appearance)
  • Agony (Leslie Gesneria) (First appearance)
  • Scream (Donna Diego) (First appearance)
• Carnage (Cletus Kasady) (Final surprise boss) (Last appearance)

Other Characters:
Some other heroes and anti-heroes aid Venom and Spidey throughout the game, making brief cameos to attack the player's foes.

• Captain America (Steve Rogers) (Last appearance)
• Ghost Rider (Johnny Blaze) (First appearance)
• Hawkeye (Clint Barton) (First appearance)
• Daredevil (Matt Murdock) (First appearance)

Locations:

• Earth-33734 (Last appearance)

Items:

• Spider-Man's Suit and Web-Shooters (Last appearance)
• The Venom Symbiote (Last appearance)
• The Carnage Symbiote (Last appearance)
• The Riot Symbiote (First appearance)
• The Phage Symbiote (First appearance)
• The Lasher Symbiote (First appearance)
• The Agony Symbiote (First appearance)
• The Scream Symbiote (First appearance)
• Captain America's Uniform and Shield (Last appearance)
• Ghost Rider's Chain
• Hawkeye's Bow and Trick Arrows
• Daredevil's Billy Club

Vehicles:

• Digger (First appearance)

Spider-Man and Venom team up against the new threat of the Life Foundation, and their five newly spawned symbiotes: Having escaped from the clutches of the treacherous Life Foundation, Venom seeks the aid of his most hated foe, Spider-Man! A tenuous alliance is formed as the Lethal Protector and the Amazing Web-Slinger race to stop the life Foundation from using the symbiotes spawned by Venom in their evil undertakings.

The player/players travel through 18 levels until the Life Foundation's plans are utterly foiled.

• City Streets
• The Bridge
• The Woods
• Enter The Foundation
• Underground City
• Mall
• Vents
• Internal Life Foundation
• Security Zone 1
• Hi-Tech Green House
• Warehouse
• Security Zone 2
• Carnage Resting Place
• Trap 1
• Trap 2
• Trap 3
• Trap 4
• Trap 5

• Despite the title of Venom: Separation Anxiety, the game mostly consists of the story comprising Venom's Venom: Lethal Protector mini-series.