Patricia and Florence is a spin-off that takes place two years after Phineas and Ferb where Patricia and Florence and their family move back to Annapolis, Maryland and what not. It also includes Buttons sub-plot where she fights Molly Collipopins.
- Patricia Verlean: The protagonist who is now 12–13 years old and is the lead singer of The Phinettes. She is the most popular girl in school in her own mind. At the end of "Join the Gal Scouts", she became troop leader of the Lassie Scouts. She is voiced by Mae Whiteman and Bridget Mendler (during Season 1 and when Mae Whiteman is voicing in the Tinker Bell movies if there's anymore left)
- Florence Verlean: The deuteragonist who is now 14–15 years old and still in sixth grade because she had to repeat pre-school because Lisa and Michael thought she couldn't talk yet. She is considered the class nerd. She is voiced by Tessa James in Season 1 then by Connie Tablot
- Buttons: Patricia and Florence's pet rabbit who is a secret agent to the O.W.C.A and fights Molly Collipopins who moved to Annapolis because the Agency hoodwinked her mom thinking that she got a job there. She is voiced by Tress MacNellie
- Haily Verlean: Patricia and Florence's adopted aged 10–11 little sister who helps them out sometimes. She is voiced by Tara Strong
- Adam Verlean: Patricia and Florence's older brother who, unlike Candace, is very lazy and sometimes dumb. He is voiced by Troy Baker.
- Alice Queenbi: Patrica and Florence's rival who is the main antagonist of the series and is always out to get Patricia and Florence. For example, when she accused Patricia of cheating on her social studies test in "Detention!" or tricking her and Florence into joining her troop The Lassie Scouts. She is voiced by Tara Strong.
- Cole Titzer: A friend of Patricia and Florence's. Often, their classmates think he and Patricia are dating. He is voiced by Moises Arias
- Brittany Frontpoke: The most mean of Alice's wannabes. She is the meanest nicknamer of the school (e.g. She calls Angela Fishler "Angel Fish", or she calls Florence "Florains", and Aqua "Auckwa"). She has a twin sister named Tiffany. She is voiced by Mae Whiteman
- Barbara Backsting: The most aggressive of Alice's wannabes who is often mean and pushy around Patricia and Florence. She is voiced by Cree Summer
- Flora "Flounder" Brightskin: A really ditzy girl who is considered by Patricia the most craziest girl she ever met. She is Goldfish Darkskull's female counterpart, but smarter. She is voiced by Jennifer Stone.
- Emile Canile: The boy who Florence was supposed to marry in "The Story of Florence Verlean" who once again tried to win Florence's heart and marry her in the future. He is the minor antagonist and is voiced by Asa Butterfield
- The Lassie Scouts: Patricia and Florence's troop while their membership of The Fireside Girls is on hold. Some of the girls included Haily, Abby, Brooke, Darby, Edith, and Jackelina. They are voiced by Tress MacNellie, Tara Strong, E.G. Daily, Cree Summer, and Ferbette.
- Toyla and Yalena: A pair of Russian witch/wizard twins introduced in "Patricia and Florence: The Movie" who have been trapped in the old house of Baba Yaga (because she mistaked them for mortal children) since the 1600s and been trapped for 400 years (4 months, 10 weeks, 10 days in their time). Toyla is voiced by Oliver Golding and Yalena is voiced by Sarah Boggler.
- Angela Fishler: Goldfish's cousin who is the smartest girl in Patricia and Florence's science class. She is voiced by Debi Derryberry.
- Principal Darlington: The principal of Susan B. Anthony Middle School where Patricia, Florence, Alice, their friends, and even their enemies attend. He is voiced by Jeff Bennet.
- Mrs. Grunker: Patricia, Florence, Alice, and Cole's homeroom teacher. She is voiced by June Foray
- Austin Fairette: An Australian pop star who Patricia has a huge crush on. He is a parody and is voiced by Justin Bieber
- Yeap: Meap's twin sister who is only a minor character.
- Merry The Platypus (Agent M) :The pet of the Verleans' next-door-neighbor and also a secret agent.
- Dr. Unbrauchbar (Un for short): Is a mad scientist and head of Unbrauchbar Evil Incorporated.
[note: the characters from the original series also make some appearances some are even minor characters like Phineas and Ferb]