| Actor/Actress |
Role |
| Shameik Moore |
Miles Morales / Spider-Man (voice) |
| Jake Johnson |
Peter B. Parker / Spider-Man (voice) |
| Hailee Steinfeld |
Gwen Stacy / Spider-Woman (voice) |
| Mahershala Ali |
Aaron Davis / Prowler (voice) |
| Brian Tyree Henry |
Jefferson Davis (voice) |
| Lily Tomlin |
May Parker (voice) |
| Lauren Vélez |
Rio Morales (voice) |
| Zoë Kravitz |
Mary Jane Watson (voice) |
| John Mulaney |
Peter Porker / Spider-Ham (voice) |
| Kimiko Glenn |
Peni Parker / SP//dr (voice) |
| Nicolas Cage |
Peter Parker / Spider-Man Noir (voice) |
| Kathryn Hahn |
Olivia "Liv" Octavius / Doctor Octopus (voice) |
| Liev Schreiber |
Wilson Fisk / Kingpin (voice) |
| Chris Pine |
Peter Parker / Spider-Man (voice) |
| Natalie Morales |
Miss Calleros (voice) |
| Edwin H. Bravo |
Brooklyn Visions Security Guard (voice) |
| Oscar Isaac |
Miguel O'Hara / Spider-Man 2099 (voice) |
| Greta Lee |
Lyla (voice) |
| Stan Lee |
Stan / Earth-67 J. Jonah Jameson (voice) |
| Jorma Taccone |
Norman Osborn / Green Goblin / Earth-67 Peter Parker / Spider-Man (voice) |
| Joaquín Cosío |
Scorpion (voice) |
| Marvin 'Krondon' Jones III |
Tombstone (voice) |
| Kim Yarbrough |
Scientist In Cafeteria (voice) |
| Lake Bell |
Vanessa Fisk (voice) |
| Nick Jaine |
Math Teacher (voice) |
| Muneeb Rehman |
Literature Teacher (voice) |
| Post Malone |
Brooklyn Bystander (voice) |
| Melanie Haynes |
Logic Teacher (voice) |
| Lex Lang |
Additional Voices (voice) |
| Christopher Miller |
Additional Voices (voice) |
| Scott Menville |
Additional Voices (voice) |
| Juan Pacheco |
Additional Voices (voice) |
| Miguel Jiron |
Additional Voices (voice) |
