Bliss possesses superhuman strength, supersonic flight, superhuman hearing, laser vision, and limited invulnerability. However, unlike Blossom, Bubbles and Buttercup and their original 1998 counterparts, her powers are uncontrollable due to her emotions. After she throws a temper tantrum that destroys the Professor's residence and leaves him unconscious, Bliss flies off to Bird Poop Island to keep everyone safe from her superpowers.

Ms. Bellum is the Mayor's loyal secretary, deputy, and the "brains behind the man", according to the Mayor himself, who believes that she really is the head of Townsville. She handles most of the Mayor's priorities, due to his general ineptness. Running gags include other onscreen characters often finding her attractive, the camera focusing on her chest, and her face always being obscured. Her name is a pun on the word "cerebellum" presumably due to her intelligent and wise nature.

Not possessing any powers or exceptional abilities, the Gangreen Gang is normally not too much of a threat to the girls, but they do delight in tormenting those weaker than themselves, especially children and the elderly.

When she is pretending to be benevolent, Sedusa's eye color is green, but when she is angry, revealing her true villainous nature, her eyes turn red.

Mojo's evil lair/observatory sits on top of an active volcano found in Townsville Central Park.

The Rowdyruff Boys are the evil male versions of the Powerpuff Girls created by Mojo Jojo using a mixture of "snips, snails, and a puppy dog tail" and toilet water in the Townsville Prison. The Rowdyruff Boys are ultimately destroyed when the girls kiss them.

Apparently, George Jetson was a citizen of Townsville as he makes a cameo in the crowd scene of the episode "Major Competition".

In the episode "Stray Bullet," the girls briefly add a fourth member, an injured squirrel who Bubbles has given Chemical X. The girls officially adopt Bullet as a Powerpuff, but Bullet feels that she must protect her animal friends and decides to go back to the woods.

Feeling overworked, the girls sneak into the lab to create a fourth Powerpuff Girl. Unlike her predecessors, Bunny is created by the girls using imitation objects of those which gave the girls life (artificial sweetener instead of sugar, dirt and twigs instead of spice, etc ...) While she possesses the same powers as the girls and a similar color theme, she is unstable and explodes in a flash of light after defeating the criminals. All that's left is a piece of her dress that falls to the ground as the girls kneel around it in mourning.

Mask Scara is a fallen fashion mogul, who vandalizes billboards, posters, and statues, putting grotesque, tacky makeup on them. She does this to the citizens of Townsville as well, making it impossible for them to remove this makeup in a desperate attempt to feel as though her trashy makeup style is still a popular trend.