Johnny Test has a long history of including a multitude of once-appearing characters. Some of these characters have made cameos in the original theme song of the series as well.
Blackbeard - Nobeard's brother and the son of Graybeard. He only cares about money and gold. With Johnny's help, he is defeated by Nobeard. This is the Blackbeard of pirate fame.
Flink - A tour guide who created a mechanical model of Pork-ness Monster in order to show it off for publicity, only to fail after it got away.
Wild Whippin' Willie - The leader of a group of motorcycle repo-men who invade the dude ranch where the Tests stay at. Johnny defeats him, however, with the help of his family and friends.
Good Johnny - A clone of Johnny created when Susan and Mary removed Johnny's bad DNA. He is uncontrollably good (as the name suggests), and this quickly becomes irritating (requisite of the time Calvin, of Bill Watterson's Calvin and Hobbes, cloned his good side).
Bad Johnny - A clone of Johnny created from a cryogenically frozen DNA sample warmed by a power outage. He is one of several Johnny clones that become uncontrollable.
Claire Nefarius - The daughter of Larius Nefarius who tried to destroy his factory. She wanted him to give her more attention, instead of working all the time. But she now has a romantic interest in Johnny Test, but now her father said that she's too young to date with him. It is unknown that will they ever meet again.
Mega Roboticle - The red robot action figure that Johnny brought to life so that Johnny could get the Nasteria action figure, He is apparently the Leader of the turbo toy force, (though he does not appear with the rest of the Turbo Toy Force in the episode Johnny's Turbo Toy Force.) Mega Roboticle parodies Optimus Prime of the Transformers series.
Nasteria - The evil dark blue robot action figure that he brought to life so Mega Robticle would have an 'exciting' battle. Mega Roboticle is Nasteria's arch enemy from the fictional series they're based on. He proved too powerful for Roboticle to defeat alone and defeated him and anyone else that attacked him. He was destroyed and returned to the toy store by The Turbo Toy Force. Nasteria parodies Zurg and Megatron.
Dr. Swampbrain - Dr. Swampbrain is Smash Badger's archenemy. He takes a hating to him and defeats him twice (the first time was in Johnny's game, and the second was when Johnny brought them to life). He is a parody of Doctor Neo Cortex, as Smash is a parody of Crash Bandicoot.
Moonsies - Creatures who live on the dark side of the moon. According to Johnny, they have brains that are shaped like one's rear end. They possess a "Cosmic Nasty Blast" maneuver. They love Super Hot Ranch Flavored Nacho Blasters.
Tiny'Mon are creatures appearing in the Johnny'Mon episodes. They parody Nintendo's Pokemon characters. There are a multitude of these creatures, and some of them are described below.
Kadoomerang - Kadoomerang is one of Blast Kechup's Tiny'mon. He is a turtle but when he evolves into Baboomerang he is a baboon.
Badias - Another of Blast Ketcuhp's Tiny'mon. He has a resemblance to Screechereen and can shoot out powerful lasers from his eyes which prove a nusiance. He seems to be based on Shadow Lugia from Pokemon XD: Gale of Darkness.
Baboomerang - The "evolved" form of Kadoomerang. He's owned by Blast Ketchup. Whenever Blast orders Baboomerang to attack, he targets Dukey.
Tree Monster - The monsters Johnny creates from trees.
Golf monster - A monster created from a golf case.
Electricity Monsters - Monsters created from power lines. The only way to defeat them is to spray water on them.
Mega Monster - When Porkbelly is invaded by monsters, the only way to stop them is to create the ultimate monster with guns, a helicopter, missiles and a fire engine. It is this weapon that wins Mr. Black and Mr. White their trip to the Fiji Islands in Johnny Test: Monster Starter.
Truck Monster - A monster created from a garbage truck.
The Unlucky Neighbor - lives across the street from Johnny. He first appeared in Johnny's Trophy Case. Usually Johnny, Susan and Mary Test cause him and his home to have bad luck, so Hugh has to pay him to cover the damages
Larius Nefarius - Larius Nefarius was the father of Clarie Nefarius. He was supposedly evil, but it was his daughter who was really the villain.
Cuddlebuns - Cuddlebuns was the weakest Tiny'Mon until Dukey/Mymuttdog showed up. He is nearly impossible to train and almost can't fly. It is Johnny's love and affection that powers him up.
Dukundra - Dukundra is Mymuttdog (Dukey)'s evolved form. He has a mace on the end of his tail. He is very strong and can launch lightening from his mouth.
Dadoomerang - Dadoomerang is Imhisdad's (Hugh's) evolved form. His "Grounding 'Rang" manuovre traps a Tiny'Mon into a inescapable dimension, literally "grounding it for life".
Road Burn - A mutant monster truck erected by Bling-Bling Boy. This due to his misinterpeting the term "monster truck". He destroys other monster trucks in order to obtain dominance, which infuriates Johnny. With Susan and Mary's help, he creates Road Burn a girlfriend, which results in a baby monster truck.
Ginger - Road Burn's wife. She mated with Road Burn and had a baby truck.
Baby Truck - The baby that Road Burn and Ginger had. He is the only monster truck that Johnny is old enough to ride.
Nobeard - The feeble pirate Johnny meets. He is underappreciated and tormented, just like Johnny is. However, when he helps Nobeard defeat his brother, he's finally treated fairly.
Greybeard - Blackbeard and Nobeard's father.
The Scream-O's - A band that was abducted by the Extreme Teen Team. They are the object of Susan and Mary's obsession.
Tiny Johnny - An inhabitant of a sub-atomic universe similar to Porkbelly which Johnny and Dukey shrink into. Other characters are also seen as inhabitants of this universe. All the characters in this universe are opposites of the real world.
Wentworth - A dinosaur Susan and Mary raised in Join the Johnny Scouts. He loves Lady Bird Scout cookies.
Bigfoot - The large-footed Canadian monster appeared in The Johnnyminster Dog Show. Susan and Mary humanize him and Johnny uses him to dominate a dog show.
Other characters include robot versions of several characters, male versions of Susan and Mary, Simeon, a used car dealer, Mr. Spurling, a client that Johnny's mom brings over for a visit, a transformed version of Gil, and others.