- Spider-Man/Iron Spider (Peter Parker) (Main story and flashback) (First appearance as Iron Spider)
- S.H.I.E.L.D. Training Program (Main story and flashback)
- Nick Fury
- Agent/Principal Coulson (Appears on a computer screen, TV, or hologram only)
- Stark Industries (First appearance)
- Living Laser (Arthur Parks) (Only appearance)
- Blizzard (Donny Gill) (Only in flashback)
- Melter (Bruno Horgan) (Only in flashback)
- Firebrand (Richard Dennison) (First appearance) (Only in flashback)
- Daily Bugle Communications
- J. Jonah Jameson (Appears on a computer screen, TV, or hologram only)
- Spider-Ham (Peter Porker) (Earth-8311) (Vision)
- Super Hero Squad (Earth-91119) (Cameo)
- Captain America (Steve Rogers) (Earth-91119) (Vision)
- Wolverine (Logan) (Earth-91119) (Vision)
- Iron Man (Tony Stark) (Earth-91119) (Cameo)
- Hulk (Bruce Banner) (Earth-91119) (Vision)
- Ms. Marvel (Carol Danvers) (Earth-91119) (Cameo)
- Thor Odinson (Earth-91119) (Cameo)
- Scarlet Witch (Wanda Maximoff) (Earth-91119) (Cameo)
- Spider-Man (Peter Parker) (Earth-91119) (Vision)
- Iron Fist (Danny Rand) (Vision) (Unknown reality)
- Power Man (Luke Cage) (Vision) (Unknown reality) (Vision)
- Spider-Man (Peter Parker) (Vision) (Unknown reality) (Vision)
- Nova (Sam Alexander) (Vision) (Unknown reality) (Vision)
- White Tiger (Ava Ayala) (Vision) (Unknown reality) (Vision)
- Earth-8311 (Cameo)
- Earth-91119 (Cameo)
- Unnamed Reality (Superheroes dancing in the disco)
- Spider-Man's Web-Shooters
- Nova Helmet
- White Tiger Amulet
- Iron Man Armor (First appearance)
- Mjolnir (Cameo)
- Iron Spider Armor (First appearance)
Spider-Man is fighting the Living Laser in the streets of New York, partially damaging some screens at the Times Square. When Spider-Man takes a distance from the villain, Laser transports himself to a neon sign near Spidey and blast it, making the hero fall, but he's caught by Luke Cage, who arrived with the rest of the trainees.
When Laser absorbs the energy of Nova's blasts he increases his size and starts a charge to fry the heroes. But Laser is attacked from above, making him expand to a point where he can't regenerate. Iron Man, the armored Avenger, flies down. Spider-Man is totally stunned as he watches his hero talking with the recently arrived Nick Fury, who wants him to stay away from S.H.I.E.L.D. business.
After Peter intervenes in the conversation suggesting a way to contain Living Laser for his obvious return, he's invited by Stark to come to the Stark Industries Main Plant. Anyways, Fury tells him to stay away from S.H.I.E.L.D. business and Spider-Man. He flies away as the trainees watch how cool he is; Fury, irritated orders them to go to the Helicarrier.
The following day, Spider-Man is walking though Stark Industries as he shows Spider-Man some inventions such as robots and a machine capable of teleporting matter to different universes; Spider-Man imagines himself in different realities.
The tour ends with Tony Stark giving to Spider-Man a new costume, at which Spidey is amazed. The next day, the S.H.I.E.L.D. trainees are at the Helicarrier waiting for Spider-Man, who isn't coming. When they finish training, he appears in the Iron Spider Armor, which he can't really control. The next week the trainees are annoyed by Spidey's new costume, which he thinks is totally value for the group and can't admit he cannot control it.
That night, Fury watches in a screen how the Living Laser's presence is detected in the city, and he sends all the trainees minus Spider-Man to battle him. When they arrive to where Laser is, Spider-Man intercepts them and tries to battle Laser, but his inexperience with the armor makes him crash across the street and damages it. Nova tries to take him down, but is ineffective as Laser absorbs his energy attacks. Laser finally, seemingly, escapes.
After the trainees leaves the scene, complaining about Spider-Man's foolishness, Living Laser takes control of the armor.
Iron Man is flying in search of Laser while he's talking with Nick Fury, who blames him for Spider-Man's recent failures thanks to his inexperience with the suit. He's suddenly attacked by the possessed Spider-Man, who alerts Tony that Laser is controlling his armor. Both battles in the sky using repulsor blasts, until Iron Man manages to go hand-to-hand and makes Spider-Man crush in the street. Laser threatens Iron Man with heating up Spider-Man's suit and kill him if he doesn't allows him to enter to his armor. Knowing there was nothing else to do, Iron Man deactivates the Firewall, but he's knocked out with one punch in the face by Spider-Man, who latter teleports to Iron Man's armor and flies away.
Later, Spider-Man is in the Helicarrier repairing his suit; when the trainees arrives and asks Spider-Man about what to do with the situation, Spider-Man states that they will fight Laser, but now he will use the armor as he could, not as he wanted.
At the Stark Industries Main Plant, Laser is trying to access top secret files in order to sell them to the highest bidder. But Iron Fist destroys the door and the trainees tries to stop him. Iron Man/Living Laser starts blasting Spider-Man, but the hero manages to doge his attacks. When the villain uses the uni-beam, his attack is neutralized by Iron Spider's blasts. Nova tries to fly to Laser, but is blasted against a wall. Taking advantage of Laser's focus on Spider-Man, Power Man brings down the villain.
Meanwhile, Spider-Man uses the data in his armor to search about Iron Man's. He finds a security failsafe, a electric discharge which could be discharged in Stark's brain, but J.A.R.V.I.S. (an AI equipped in the armor) suggests not to do it.
Back to the fight, Power Man is battling hand-to-hand with Laser, and the villain manages to stop a kick from Cage and throw him to Nova. White Tiger tries to hit him, but her attacks are blocked, after she dodges a blast from Laser, he knocks her with the uni-beam. Iron Fist also tries, but is thrown back by the armor's repulsor boots. Then, Laser uses missiles to attack the heroes, Power Man and White Tiger eludes them, and Iron Fist protects he and Nova by lifting the floor with a punch and taking them the rest with his iron fist.
Spider-Man confronts the Laser and activates the failsafe, waking up Tony, who reactivates the firewall and expels Living Laser from his armor. Then, Peter suggest Iron Man to use the teleporting machine to get rid from Laser. After making him increase Laser's size, Iron Man flies away, making the villain to chase him around Stark Industries. Meanwhile, Spider-Man gets to the teleporter controls along the trainees; via communicator, Iron Man asks Spider-Man to turn on the machine at his signal, but Spidey doesn't feel he can do it.
When Tony finally makes Laser enter the machine, he gives Spider-Man the signal, at what the hero proceeds to turn it on. When the situation is over, the heroes asks themselves to which dimension could Laser be; at that moment, he arrives to the Earth-91119 and is attacked by that reality's Thor. Back to Stark Industries, Spider-Man explains Stark that the Iron Spider Suit is great, but he isn't ready to control it.
At the Helicarrier, Peter apologizes to his teammates; suddenly, Nick Fury arrives to the room where they are and makes everyone but Spider-Man to get out. Fury leads Spider-Man to a armory, where he shows him a upgraded Iron Spider suit which can turn into a backpack. Peter wants to use it again, but when he suits up, he ends up disordering all the room. Fury smiles and walks away, leaving Spider-Man to order the mess he made.
- This episode features a "Fury Files" mini-clip of Iron Fist during the credits.
- The universe where Living Laser ends up is where The Super Hero Squad Show takes place.
- This episode is a company cross-over contradiction because Ultimate Spider-Man takes place on Disney XD while the Super Hero Squad Show took place on Cartoon Network first and then, on the Hub (now Discovery Family). Both of these series however, are available on Disney+.