| Shameik Moore | | Miles Morales (voice) |
| Jake Johnson | | Peter B. Parker (voice) |
| Hailee Steinfeld | | Gwen Stacy (voice) |
| Mahershala Ali | | Uncle Aaron (voice) |
| Brian Tyree Henry | | Jefferson Davis (voice) |
| Lily Tomlin | | Aunt May (voice) |
| Luna Lauren Velez | | Rio Morales (voice) |
| Zoë Kravitz | | Mary Jane (voice) |
| John Mulaney | | Spider-Ham (voice) |
| Kimiko Glenn | | Peni Parker (voice) |
| Nicolas Cage | | Spider-Man Noir (voice) |
| Kathryn Hahn | | Doc Ock (voice) |
| Liev Schreiber | | Wilson Fisk (voice) |
| Chris Pine | | Peter Parker (voice) |
| Natalie Morales | | Miss Calleros (voice) |
| Edwin R. Habacon | | Brooklyn Visions Security Guard (voice) |
| Oscar Isaac | | Interesting Person #1 (voice) |
| Greta Lee | | Interesting Person #2 (voice) |
| Stan Lee | | Stan (voice) |
| Jorma Taccone | | Green Goblin / Last Dude (voice) |
| Joaquín Cosio | | Scorpion (voice) |
| Marvin Jones III | | Tombstone (voice) |
| Kim Yarbrough | | Scientist In Cafeteria (voice) |
| Lake Bell | | Vanessa Fisk (voice) |
| Jessica Mikayla | | Brooklyn Friend (voice) |
| Gredel Berrios Calladine | | Brooklyn Friend (voice) |
| Sarah D. Cole | | Brooklyn Friend (voice) |
| Kelby Joseph | | Brooklyn Friend (voice) |
| Mimi Davila | | Brooklyn Visions Student (voice) |
| Claudia Choi | | Brooklyn Visions Teacher (voice) |
| Melanie Haynes | | Brooklyn Visions Teacher (voice) |
| Joseph Izzo | | Brooklyn Visions Teacher (voice) |
| Nick Jaine | | Brooklyn Visions Teacher (voice) |
| Muneeb Rehman | | Brooklyn Visions Teacher (voice) |
| Carlos Zaragoza | | Brooklyn Visions Teacher (voice) |
| Post Malone | | Brooklyn Bystander (voice) |