Many characters in the animated series 64 Zoo Lane are portrayed as heroes in many episodes. Some of them are not considered of the villains, but some are seen portrayed with the villainous roles. This list doesn't include such with the characters such as Victor or the other villains encountered in the series.
- The Story of Beverly the Beaver: Refuses to share her popcorn with the monkeys Giggles and Tickles as Georgina isn't happy what Lucy did to the monkeys.
- The Story of Nelson the Elephant: As a calf, Nelson disturbs Nathalie, Audrey and Reginald and refuses to stop jumping after the animals keep on giving him a warning.
- The Story of Giggles and Tickles: They play a trick on the animals while waiting near a large rock and the animals have had enough of their tricks.
- The Story of Petula the Parrot: They both pull a branch (equivalent to a lever) that ejects Petula's fruits and Petula becomes enraged after the 2 monkeys start eating her fruit, even without asking permission from her.
Though grumpy, Reginald is not considered a villain, but he is seen doing antagonistic roles.
- The Story of Herbert the Warthog: Tries to stop the music and noise created by Herbert, Alan, Zed and the 3 frogs. He later reforms when an audience of the animals tell him to participate in the concert.
- The Story of Reginald's Big Sleep: Gets upset with all the animals and tells them to leave him alone. After disturbed by several insects, Reginald is bothered by many of them and retreats from the grass. After reforming of how selfish he has been, the other animals start to clear away many of the grass, but Reginald tells them to leave the last of the grass for some of the insects.
- The Story of Seamus the Stork: He is jealous of Seamus and his flying activities. Thus, he shows no respect after Seamus loses his feathers, while the other birds feel bad for the loss of his feathers. He later reforms with the other birds after Seamus had the ability to fly again.
Sometimes bossy, Doris is not considered a villainess. However, she is also seen doing antagonistic roles.
- The Story of the Dawn Chorus: After being bothered by a dawn chorus, Doris decides to make her own chorus and tries to let all the animals copy the sound of a quacking duck. However, the animals refuse to listen to Doris, since not all of them are the ducks like Doris and not all of them can quack like a duck.
Jimmy has only been a villain to Joey once in one episode. Since Episode 20, he reforms with Joey and Phoebe.
- The Story of Joey the Kangaroo: Taunts Joey that he is unable to jump for the event where the young kangaroos must learn how to jump and to see who jumps the highest. He taunts Joey that he cannot beat him in the jumping contest.
- The Story of Zed the Zebra: Taunts the other animals to lose the race, because he tells them he's very fast with his stripes. However, the other animals win the race instead and Zed doesn't feel bad after he loses.
- The Story of Gary's Best Friend: Loses his temper after Gary (in his zebra disguise) thinks Zed is vain. By losing his temper, Zed yells at Gary rudely and Georgina, Molly and Nathalie get upset at him for being rude to Gary. He later does apologize for being rude to Gary and Gary teaches him how to dance with him.
- The Story of Herbert's Peaceful Day: Disturbs the animals with his noisy behavior, such as bothering Reginald with his musical rocks, talking to himself rudely and eating very noisy that disturbs Doris. This causes 3 of them to give a challenge to see if he remains silent for one day until sunset to get a big watermelon and if he fails, he won't receive the watermelon.
- The Story of Pauline the Pelican: Steals a fish from Seamus, a watermelon from Herbert and a bone from Reginald without asking permission. This causes them to be upset at Pauline, even though she was hungry and unable to catch a fish.
- The Story of Melanie the Moose: Gets upset at Melanie by yelling at her for being clumsy and useless, along with Boris and Beverly. Later, he, Boris and Beverly apologize for being rude to her.
- The Story of Melanie the Moose: Gets upset at Melanie by yelling at her for being clumsy and useless, along with Boris and Randolph. Later, she, Boris and Beverly, apologize for being rude to her.
- The Story of Beverly the Beaver: Cuts down almost all the trees to build a big dam and the other animals are not happy for her selfish behavior. This is Beverly's most antagonistic role.
The Other unnamed animals
- The Story of Henrietta the Hairy Hippo: Some of the hippos taunt Henrietta when they see her hairy body and the protagonist of the story doesn't feel happy when the others taunt her. They later reform when they see her beautiful hairdo done by the Snip Snip Bird.
- The Story of Reginald's Big Sleep: Several insects begin to bother Reginald who is sleeping in the tall grass. Reginald was able to escape from the noisy grass. This was a misunderstanding for all the animals, but the insects never show remorse to Reginald, as none of them are seen when the animals are clearing away the tall grass.