 Shameik MooreMiles Morales (voice) | Miles Morales (voice) |
 Jake JohnsonPeter B. Parker (voice) | Peter B. Parker (voice) |
 Hailee SteinfeldGwen Stacy (voice) | Gwen Stacy (voice) |
 Mahershala AliUncle Aaron (voice) | Uncle Aaron (voice) |
 Brian Tyree HenryJefferson Davis (voice) | Jefferson Davis (voice) |
 Lily TomlinAunt May (voice) | Aunt May (voice) |
 Luna Lauren VélezRio Morales (voice) | Rio Morales (voice) |
 Zoë KravitzMary Jane (voice) | Mary Jane (voice) |
 John MulaneySpider-Ham (voice) | Spider-Ham (voice) |
 Kimiko GlennPeni Parker (voice) | Peni Parker (voice) |
 Nicolas CageSpider-Man Noir (voice) | Spider-Man Noir (voice) |
 Kathryn HahnDoc Ock (voice) | Doc Ock (voice) |
 Liev SchreiberWilson Fisk (voice) | Wilson Fisk (voice) |
 Chris PinePeter Parker (voice) | Peter Parker (voice) |
 Natalie MoralesMiss Calleros (voice) | Miss Calleros (voice) |
 Edwin H. BravoBrooklyn Visions Security Guard (voice) | Brooklyn Visions Security Guard (voice) |
 Oscar IsaacInteresting Person #1 (voice) | Interesting Person #1 (voice) |
 Greta LeeInteresting Person #2 (voice) | Interesting Person #2 (voice) |
 Stan LeeStan (voice) | Stan (voice) |
 Jorma TacconeGreen Goblin / Last Dude (voice) | Green Goblin / Last Dude (voice) |
 Joaquín CosíoScorpion (voice) | Scorpion (voice) |
 Marvin Jones IIITombstone (voice) | Tombstone (voice) |
| KKim YarbroughScientist in Cafeteria (voice) | Scientist in Cafeteria (voice) |
 Lake BellVanessa Fisk (voice) | Vanessa Fisk (voice) |
 Jessica Mikayla AdamsBrooklyn Friend (voice) | Brooklyn Friend (voice) |
| GGredel Berrios CalladineBrooklyn Friend (voice) | Brooklyn Friend (voice) |
 Sarah D. ColeBrooklyn Friend (voice) | Brooklyn Friend (voice) |
| KKelby JosephBrooklyn Friend (voice) | Brooklyn Friend (voice) |
 Mimi DavilaBrooklyn Visions Student (voice) | Brooklyn Visions Student (voice) |
 Claudia ChoiBrooklyn Visions Teacher (voice) | Brooklyn Visions Teacher (voice) |
