Peter Parker discovers his life is a lie, as he discovers another person having his name.
- Peter Parker/Spider-Man (Jason Marsden) - the real one, who was incarcerated on a Skrull ship.
- Peter Parker/Spider-Man/Ben Riley (Tom Holland) - The Clone of the Original Peter Parker, who discovers his life is a lie when he sees Black Widow disguised as Aunt May, he was made to find the Spider-Sense in his DNA. He was the Second Result of the Cloning Program.
- Mary Jane Watson (Emmanuel Chirqui) - Peter's wife who realizes the clone and reports it the Peter.
- Harry Osborn (Kevin Miller) - ???
- Flash Thompson (Matt Mercer) - ???
- Liz Allan (Natalia Cigliuti ) - ???
- Felicia Hardy/Black Cat (Sumalee Montano) - ???
- Maxwell Dillon/Electro (Crispin Glover) - reforms and aids Spider-Man to stop the Skrulls.
- T’Challa/Black Panther (Kevin Michael Richardson) - King of Wakanda and studying the Spider that mutated him.
- Ororo Munroe/Storm (Gina Torres) - Queen of Wakanda and a friend of the Parker Family.
- Luke Cage (T.C. Carson) - hired by S.H.I.E.L.D to apprehend the Chameleon.
- Natasha Romanoff/Black Widow (Kimberly Brooks/Candi Milo in Aunt May disguise) - she posed as Aunt May to keep the clone from discovering he’s a clone, She was later revealed to be the Queen Skrull Veranke.
- Clone Experiments:
- Kaine (Andrew Garfield) - first result, until the skrulls infiltrated and intervened with the final results, he survived the explosion to be scarred.
- Spidercide (Toby McGuire) - third result, the third clone created in Hydra.
- Dmitri Smerdyakov/Chameleon/Gustov Fiers (Alan Tudyk) - the film’s main antagonist, orchestrated the Skrull Electro for a skin sample, his goal is to get revenge on Spider-Man for his disguise failure as him, he is responsible for the Skrulls getting access to S.H.I.E.L.D.s cloning program and the Man in the Shadows.
- Kraven the Hunter (Luke Evans) - the film’s secondary antagonist, hunting Spider-Man and his brother for his foolish business with the Skrulls.
- Lonnie Thompson Lincoln/Tombstone (David Owylewo) - the film’s tertiary antagonist, a gang leader who puts a price on Spider-Man.
- HYDRA: the film’s quaternary antagonists, after the cloning program.
- Silvermane (John Malcovich) - the head of Hydra in the New York providence.
- The film is rated PG for Language and Brief Nudity
- Differences from the comic and the film:
- It was revealed Electro from Amazing Spider-Man 2 was a Skrull and Chameleon orchastrated it for a skin sample.
- Character designs are done by Jeff Matsuda, for his work on The Batman.