Michael Wilson / The Legendary Spider-Man - The protagonist, a seasoned and heroic Spider-Man from the 1947 universe who leads the team of Spider-People.
Miles Morales / Spider-Man - The deuteragonist, a teenager from the present day who gains spider-like abilities and becomes a new Spider-Man, mentored by Michael Wilson and Peter B.Parker.
Peter B. Parker / Spider-Man -The Tritagonist, An older, jaded version of Spider-Man from another dimension who assists Michael and Miles.
Gwen Stacy / Spider-Woman (Spider-Gwen) - A skilled and confident Spider-Woman from another dimension.
Spider-Man Noir - A black-and-white, hard-boiled detective version of Spider-Man from the 1930s.
Peni Parker / SP//dr - A young girl from a futuristic dimension who pilots a spider-robot.
Peter Porker / Spider-Ham - A cartoonish, anthropomorphic pig version of Spider-Man.
Wilson Fisk / Kingpin - The main antagonist, a powerful crime lord who seeks to bring back his family from another dimension.
Aaron Davis / Prowler - Miles' uncle who works for Kingpin but has a complicated relationship with Miles.
Jefferson Davis - Miles' father, a police officer who initially disapproves of Spider-Man.
Rio Morales - Miles' supportive mother.
Livia Octavius / Doctor Octopus - A female version of Doctor Octopus who works for Kingpin.
Aunt May - The aunt of Peter Parker who assists Michael, Miles, and the other Spider-People.
Mary Jane Watson - Peter B. Parker's ex-wife from his dimension.
Spider-Man (Earth-1610) - The original Spider-Man in Miles' universe who dies early in the film.
Green Goblin - A monstrous version of the Green Goblin who works for Kingpin.
Scorpion - Another villain working for Kingpin.
Tombstone - Kingpin's enforcer..