A super-powered mouse who always saves the day and protects innocent mice from cats and other felons.
- Alliterative Name: Both parts of his name begin with M. This also applies to Mike Mouse, the name he assumes for his civilian identity in Mighty Mouse: The New Adventures.
- Captain Ersatz: He's heavily based on Superman. He even wore blue and was called Supermouse in the earlier shorts.
- Catchphrase: He often announces that he'll spring into action by singing "Here I come to save the day!"
- Intergenerational Friendship: He is friends with an orphan boy named Scrappy in Mighty Mouse: The New Adventures.
- Nice Guy: He's quick to defend his fellow mice from danger.
- Pintsized Powerhouse: He's small like any other mouse, but is still strong and formidable enough to go toe-to-toe with every enemy he faces.
Mighty Mouse's main love interest.
- Alliterative Name: Her first name and her last name both begin with the letter P.
- Damsel in Distress: Her usual role is to be in peril so that Mighty Mouse can save her.
- Formerly Fit: The Mighty Mouse: The New Adventures episode "Mighty's Wedlock Whimsy" has Pearl become tremendously obese in Mighty Mouse's dream of being married to her.
- Graceful Ladies Like Purple: She wears a purple dress most of the time.
- Reused Character Design: Some of the theatrical shorts have Mighty Mouse instead save an identical mouse woman named Nell.
Oil Can Harry
Mighty Mouse's main foe, who often tries to pursue Pearl Pureheart.
- Abhorrent Admirer: He's in love with Pearl Pureheart, but she doesn't want to be with him.
- Arch-Enemy: He is Mighty Mouse's most prominent adversary.
- Cats Are Mean: He's a cat and evil.
- Dastardly Whiplash: He resembles and acts like the standard 1920's serial villain with a moustache and fancy clothes.
- Demoted to Extra: He's Mighty Mouse's most prominent adversary in the theatrical shorts and the Filmation series, but he only appears in two episodes of Mighty Mouse: The New Adventures, not counting episodes that recycle footage from the theatrical cartoons.
- Evil Sounds Deep: His voice is very deep, though less so in the Filmation cartoon and Mighty Mouse: The New Adventures.
- Fat and Skinny: He becomes part of this sort of pairing in the Filmation series, which contrasts his being a skinny and competent villain by giving him an overweight and bumbling henchman named Swifty.
- Lean and Mean: He's tall, thin, and not very nice.
- Rogues Gallery Transplant: While better known as a Mighty Mouse adversary, Oil Can Harry got his start as a human villain in Terrytoons' much older Fanny Zilch cartoons.
- Sanity Slippage: The Mighty Mouse: The New Adventures episode "Still Oily After All These Years" shows that his fixation on Pearl Pureheart over the years has driven him mad.
A cat with a bald spot who is completely nuts and out to cause havoc.
- Alliterative Name: His first and last names both start with the letter P.
- Ax-Crazy: He is frequently shown to be mentally unstable.
- Bald of Evil: He has no hair on top of his head.
- Berserk Button: The most common way to set him off is to say his name out loud in front of him.
- Captain Ersatz: He is most likely a spoof of Superman's archenemy Lex Luthor, particularly since he's a bald supervillain whose name is an alliteration.
- Cloudcuckoolander: He sometimes shows bizarre habits, like when he gives himself a tan at the end of his debut episode solely so he can look good for his picture as he's taken to jail.
- No Indoor Voice: Nearly all of his dialogue has him yelling at the top of his lungs like the deranged maniac he is.
- Paper-Thin Disguise: In "Day of the Mice", he disguises himself as a mouse by wearing Mickey Mouse ears.
- You're Insane!: In his debut in "Night on Bald Pate", Pearl Pureheart responds to his boasting of scheming to steal every left shoe in Mouseville by calling him mad.
An orphan mouse who is friends with Mighty Mouse.
- Demoted to Extra: His appearances become more scarce in the second season, the only episodes of that season featuring him being "Anatomy of a Milquetoast" (which lampshades his sudden absence by having Mighty Mouse on trial because he's accused of being responsible for Scrappy's disappearance) and "Mighty's Tone Poem" (where he makes a cameo).
- Half-Dressed Cartoon Animal: The only clothes he wears are pants.
- Intergenerational Friendship: He's a child who is friends with the adult Mighty Mouse.
- Secret Keeper: "All You Need is Glove" has Mighty Mouse reveal his secret identity to Scrappy and swear him into secrecy.
A bat-themed superhero who happens to be a bat himself.
- Captain Ersatz: He's an obvious Batman pastiche.
- Department of Redundancy Department: He's a bat who fights crime as a bat-themed superhero named Bat-Bat.
- Lantern Jaw of Justice: He has a big chin.
A villainous cow who wants to make everyone stop using dairy products.
- Animal Gender-Bender: He's a cow, but is male.
- HeelFace Turn: He reforms and becomes friends with Mighty Mouse in Mighty's dream of being married to Pearl Pureheart in "Mighty's Wedlock Whimsy" as well as the future shown in "The Bride of Mighty Mouse".
- No Indoor Voice: He yells pretty much every time he talks.