Spider-Man – The titular hero of the series, Spider-Man is weaker and faster than his teammate, Venom. As in the comic, he is able to punch, kick, swing from webs and cling to walls. In combat, his special moves are oriented toward agility and finesse rather than brute strength.
Venom – As a symbiote, Venom is a combination of both human and alien organism. He has all of Spider-Man's generic abilities (web-swinging and wall-crawling), but unlike Spider-Man, his combat moves are primarily based on raw power. He also takes less damage per hit than Spider-Man.
Carnage- The main antagonist of the game, and appears as the final boss.
The Jury – Unlike the Symbiotes, these level bosses are not superhuman; instead, they are extremely well armed and well equipped mercenaries. They appear singly and in pairs at the end of selected levels, and again together at the end of the game. The members are Sentry, Ramshot, Screech, Bomblast, Firearm and Wysper.
Symbiotes – Like the Jury, these hybrid alien/human level bosses possess varied combat skills and attack either alone or in pairs until the end of the game. The symbiotes are Riot, Agony, Lasher, Scream and Phage.
Though the game is titled after the comic book series Venom: Separation Anxiety, the story more closely follows the events of Venom's first limited series, Venom: Lethal Protector. The game's plot loosely follows Lethal Protector in the following ways:
Appearance of Spider-Man (in Lethal Protector #1.)
Confronting the digger in San Francisco (as seen in Lethal Protector #1.)
Discovering the underground city (as seen in Lethal Protector #1.)
Appearance of The Jury (as seen in Lethal Protector #2.)
Removal of five symbiote seeds to create five new symbiotes (as seen in Lethal Protector #4.)
Spider-Man and Venom's escape from the Life Foundation (as seen in Lethal Protector #5.)
Confronting the five symbiotes at the Life Foundation Headquarters (as seen in Lethal Protector #5.)