Dr. Harlow is a semi-dark orange muntjac deer.[1] Wrapped around his neck is a black stethoscope. Underneath his coat is a blue medical scrub with a red tie. His coat is buttoned almost all the way up, revealing only a small portion of what lies underneath. On his head rests a head mirror with a brown band. In his right hand, he carries a black suitcase with the words "TOP SECRET" in red letters on it, similar to Ron from Accounting's suitcase. His face features slit eyes with black, shiny, irises, and his mouth is covered by a blue surgical face mask. On top of his head are two large, grey antlers. His forehead has dark orange patterns on his face, and his ears are a slightly darker color than his face. Little is known about Dr. Harlow's personality. As revealed in his hospital lobby dialogue, he has worked with the Animal Hospital for a long time and has seen many hires come and go. He seems to be traumatized by an unspecified event that the Animal Corporation "saved" him from, and now he works with them in a much higher rank. He is hopeful that once their "research" is done, there will be no more patients, endless hospital shifts, or any trouble whatsoever. Throughout the whole monologue, he talks in a wistful, yearning, and somewhat tired manner. The only things Dr. Harlow can be seen doing are sitting in the lobby while watching the player(s) do all the work, speaking with an Animal Corporation employee in a hazmat suit outside the hospital, going to the staff room, or coming to the reception office after the shift has ended.

The Secret Agent is a fully black, cat-like creature with pointy ears, cheek fur and black whiskers. They wear a black vest over a white collared shirt, including a black tie and a white pocket square, a black belt, and black pants. Their face features slit eyes with a vibrant yellow sclera resembling a cat's eyes, and their mouth and nose appears to be invisible, unless behind a glass door. They are seemly to be strict, fair, strict on rules, and supervised by Dr. Harlow.

The Head Nurse is a creature of an unknown species with gray spots and pointed ears or horns. They wear light blue medical scrubs, with a large pocket on each lower side of the shirt. Hanging from their neck is a black stethoscope. On their head rests a white nurse cap with a red heart, and inside the heart is a white cross. Their face features large, half-lidded round eyes with prominent black eyelids and a small pout. Meanwhile, she is serious, fair, and is strict in her workplace.

The Intern is a rabbit-like creature with upturned ears. It wears a white jacket hoodie/coveralls with grey pockets, a zipper and black drawstrings, and plain white pants. Their face features small, black dots as eyes, and a large, uncanny smile. They are light orange in the classes shop and by default, but varies in color once in a shift. When playing with other users, Intern can appear green, blue, or pink. They are silly, nervous, shy, but also hard working!

Barney appears as a white-furred rabbitoid creature with narrow black eyes and a thick mustache. He wears a burlap brown beret, along with plaid brown pants. His Anomaly form has a large sharp-toothed mouth, as well as wide, hollow pitch-black eyes with small white pupils. He also brandishes the Scalpel given to him by the player.Barney Animal Hospital Wiki 93 Barney initially comes across as courteous, mannered, kind, and respectful in his early interactions. Every time he receives help from or his favors are fulfilled by the player, he either thanks you or compliments the player in some generous way (ex: "You are the best..."). Almost every message from Barney includes him calling the player a friend. Despite this, he uses the player as much as possible and exploits them to give him what he wants, especially coffee, and frankly appears as very demanding and manipulative—a side that rears its head when he's denied—as he gets upset and even makes threats if the player refuses to give him what he asks for. He is heavily dependent on the player for favors.

Ratthew is a small black rat. He has a white snout, two white buckteeth, and a long, curled pink tail. Oddly, his ears resemble those of a rabbit more closely than a rat's. Sometimes, when he appears, he will stare at the camera, but will not turn his head more than physically possible.Ratthew comes off as paranoid but kind-hearted. He seemingly has a lot of distrust that stems from past experiences. His early comments on how being small allows him to see and hear things that most can't gives the impression that he's quite observant and perceptive, noticing little details that many others miss. Ratthew hiding his identity during the first interaction makes him come off as somewhat secretive. He appears quite cautious, as he warns the player not to trust anyone, especially those carrying suitcases, and he later tells the player he has to go into hiding