Mrs. Frisby is a widowed field mouse and the devoted mother of four children living in a cinder block on the Fitzgibbon farm. Though naturally timid, she possesses a deep, maternal courage that drives her to face dangerous predators and mysterious animals to save her family. Her lack of knowledge regarding her late husband's past leaves her initially unaware of the larger forces operating around her.

Nicodemus is the dignified leader of the highly intelligent colony of rats living beneath the rosebush. He carries a satchel and bears a scar over his left eye from an encounter with the farm cat. He holds a strong desire to lead his people away from their reliance on human resources and establish an independent, self-sustaining society in the wild.

Justin is a highly intelligent and courageous rat who serves as one of the key members of the colony. He approaches dangerous situations with a lighthearted but focused demeanor, regularly volunteering for the most perilous tasks. His natural bravery makes him instrumental in the rats' operations and their interactions with the outside world.

Mr. Ages is an elderly white mouse who creates medicinal draughts and powders from herbs, seeds, and mushrooms. He possesses an unusually long lifespan and a sharp intellect, making him a highly respected figure among the animals on the farm. He holds a close connection to the rats of the rosebush and provides crucial assistance to the Frisby family.

Dragon is the enormous, fiercely predatory orange-and-white farm cat who patrols the Fitzgibbon property. He acts as a constant, lethal threat to all the rodents and birds on the farm, forcing them to meticulously plan their movements around his sleeping habits.

Jenner is Nicodemus's childhood best friend and a prominent figure among the rats. He is highly intelligent but harbors deep disagreements with Nicodemus regarding the colony's moral direction and lifestyle. He questions the necessity of giving up their comfortable, luxurious life in favor of a harder, independent existence.

Jonathan Frisby is Mrs. Frisby's late husband, a mouse who possessed remarkable intelligence and taught his children how to read. Though the farm cat killed him before the events of the story, his legacy commands immense respect from the forest's most powerful creatures. He kept many secrets from his wife to protect her from the burdens of his unusual past.

Timothy is the youngest of the Frisby children, characterized by his bright eyes and deep thoughtfulness. He is the frailest of his siblings, having suffered a mysterious illness as an infant, and his sudden bout of pneumonia sets the story's events in motion. He closely resembles his late father in both intellect and his tendency to worry.

The Owl is the oldest and most respected animal in the forest, serving as a silent observer and keeper of knowledge. He is an imposing, predatory bird who typically ignores mice, but he makes an exception for Mrs. Frisby out of respect for her late husband. He views the changing world from his dying tree, refusing to leave his long-time home.

Jeremy is a young, somewhat foolish crow with a fondness for collecting shiny objects. Despite his lack of common sense, he is fiercely loyal and grateful to those who help him. He provides crucial transportation and information to Mrs. Frisby, acting as her guide to the wider, more dangerous world beyond the garden.

Brutus is a massive, muscular young rat who stands guard at the stone archway leading into the rats' underground domain. He is dutiful and strictly follows orders, intimidating outsiders who attempt to enter the tunnel system without proper authorization.

Teresa, Martin, and Cynthia are the older siblings of the Frisby family. Teresa is the eldest and helps care for the others; Martin is the largest and most headstrong son; and Cynthia is the youngest daughter, noted for being slim and optimistic. Together, they form the emotional anchor that drives Mrs. Frisby's desperate quest to save their home.

Mr. Fitzgibbon is the farmer who owns the land where the mice and rats reside. He dictates the schedule of the farm, and his routine agricultural work poses a massive, existential threat to the tiny creatures living in his garden.

Dr. Schultz is a scientist working at the NIMH laboratory, focused on testing the effects of specialized injections on rodent intelligence. He observes the rats closely but underestimates the extent of their comprehension and capability.

Billy is the younger son of the Fitzgibbon family. He is observant and quick, making him a sudden, unpredictable danger to the small animals that travel through the farmhouse.

Isabella is a young female rat living in the rosebush colony. She is initially suspicious of outsiders but possesses a talkative nature, inadvertently revealing pieces of information about the colony's origins and internal disagreements to Mrs. Frisby.

Paul is the older son of the Fitzgibbon family. He assists his father with the farm labor, contributing to the human activity that threatens the smaller creatures of the fields.

Mrs. Fitzgibbon is the farmer's wife who manages the household. Her presence in the kitchen dictates the schedule for when the animals can safely approach the cat's food bowl.

Gerald is Nicodemus's older brother from their time living as ordinary rats in a marketplace. He represents the simple, unenhanced life the rats led before their capture.