* Venom: The main antagonist of the film, created when an alien symbiote bonds with Eddie Brock.
* The Sandman: A villain created by the powers of the sand, who is seeking revenge against Spider-Man for the death of his brother.
* The New Goblin: Harry Osborn, seeking revenge for the death of his father (the original Green Goblin), who was killed by Spider-Man.
* The Green Goblin: A short appearance by Norman Osborn, who appears as a hallucination of Peter Parker's guilt over the death of his father.
While not necessarily a "villain" in the traditional sense, the symbiote suit itself also poses a significant threat to Peter Parker, as it amplifies his aggression and darkness, tempting him to embrace the darker aspects of his personality.