When Professor Utonium set out to create the perfect little girl, he brewed up a concoction that included sugar, spice, and everything nice. Then he accidentally added "Chemical X" to the mixture and the Powerpuff Girls were born.

Mojo Jojo was originally Jojo, the Professor's hyperactive lab monkey. During the creation of the Powerpuff Girls, Jojo pushed the Professor towards a container of Chemical X, causing it to break and spill into the concoction. This resulted in an explosion, which not only created the Powerpuff Girls, but also caused Jojo's simian DNA to mutate, giving him super-intelligence.

The girls all live in the fictional city of Townsville, which is famous for crime rates, giant monster attacks, day-savings, Little Tokyo district, and taffy. The girls are frequently called upon by the city's mayor to help fight crime.

Although he is generally referred to only as "Mayor", he is referred to as "Barney" by his wife in the episode "Boogie Frights".

Blossom is the tactician and self-proclaimed leader of the Powerpuff Girls. Her personality ingredient is "everything nice", and her signature color is bright pink.

Ms. Keane forbids any fighting in school, even if there is a crisis taking place. She teaches the girls that there are other ways to solve problems besides fighting as demonstrated in the episodes "Schoolhouse Rocked" and "Imaginary Fiend".

The Talking Dog is abused in almost every appearance he makes, but, as a running gag, no one ever seems to notice his suffering. Most of the time these incidents are caused by the Powerpuff girls accidentally hitting him or crushing him under some large object.

In the episode "Moral Decay", Buttercup accidentally knocks out one of Bubbles' teeth. The next day when she discovers a dollar from the Tooth Fairy under Bubbles' pillow, Buttercup sees an opportunity to make money by knocking out bad guys' teeth and putting them under her pillow.

Mitch Mitchelson torments the kids in the class in a few episodes, though he is not a major threat. He is Buttercup's close friend, according to Buttercup in her interview in the DVD features of The Powerpuff Girls Movie. For a brief time he hosted a short segment on Cartoon Network called "Mitch Rocks", where he looked at various things and said whether they "rock" or not.

While Bubbles has a number of powers, including animal whispering, multilingualism, thunder claps, and laser-eye beams, it is Blossom who has a photographic memory.