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.

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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..