- Spider-Man (Peter Parker)
- Agent Venom / Venom / Scarlet Spider (Flash Thompson) (1st and only appearance as Scarlet Spider) (Merges with the Venom Symbiote) (Joins S.H.I.E.L.D. and S.H.I.E.L.D. Trainees)
- Harry Osborn
- Daily Bugle
- J. Jonah Jameson (Appears on a computer screen, TV, or hologram only)
- Mary Jane Watson
- Cloak (Tyrone Johnson) (Appears on a computer screen, TV, or hologram only)
- Dagger (Tandy Bowen) (Appears on a computer screen, TV, or hologram only)
- Gravity (Greg Willis) (Appears on a computer screen, TV, or hologram only)
- Speedball (Robbie Baldwin) (Appears on a computer screen, TV, or hologram only)
- Ka-Zar (Kevin Plunder) (Appears on a computer screen, TV, or hologram only)
- Squirrel Girl (Doreen Green) (Appears on a computer screen, TV, or hologram only)
- Triton (Appears on a computer screen, TV, or hologram only)
- Echo (Maya Lopez) (Appears on a computer screen, TV, or hologram only)
- Avengers (Mentioned)
- Loki (Mentioned)
- Phil Coulson (Mentioned)
- Rock Trolls (Mentioned)
- S.H.I.E.L.D. Trainees (Mentioned)
- Beetle Armor
- Spider-Man's Web-Shooters
- Scarlet Spider's Staff
- Taskmaster's Shield
- Taksmaster's Sword
Spider-Man hunts throughout New York for the last remnants of Venom. He finds a piece of one hang off one of J. Jonah Jameson screens. As he extracts the piece he is attacked by the Scorpion who is possessed by another piece of Venom. While fighting the Venomized Scorpion, Flash Thompson appeared, wearing a Scarlet Spider costume and calling himself Scarlet Spider. Spider-Man told Flash not to be a hero and defeated Scorpion, unaware that a small piece of Venom attached to Flash. The next day, the Beetle, under orders from Taskmaster, tracked Venom to Midtown High School and chases Flash. Spider-Man stashes Flash into a locker to protect him, but Flash wanted to help. The suit activated, and Flash battles the Beetle. When Flash has him subdued, Spider-Man tries to talk Flash into giving up the suit, and the two fight until Taskmaster arrived. Venom and Spider-Man battled the Beetle and Taskmaster, Flash integrated parts of Beetle's suit, including rocket launchers and armor, and fully becoming Agent Venom. After the fight, Agent Venom relents that being a hero is harder than he thought and tries to get the suit to leave him. However, the suit has found a perfect host in Flash and permanently bonded to him. Spider-man convinces Fury to take him on as a member of the team. As he reveals his actions has encouraged other young heroes to come out of the wood work.
- The third season of Ultimate Spider-Man was aired on Disney XD UK before its August release date in the US.
- Scorpion bonding with the symbiote could've been a reference to the Earth-616 Scorpion having become Venom for years.
- Flash originally wears a make-shift Scarlet Spider costume.
- This is the first animated adaptation of Agent Venom.
- During the third season, Spider-Man doesn't meet Gravity, Speedball and Echo.