The unnamed narrator is a young man from a wealthy background who travels to a remote, decaying estate after receiving an urgent letter from a boyhood friend. Highly sensitive to his environment, he relies on rationality and factual observation to process the eerie atmosphere of the house and the strange condition of its inhabitants. He acts as an objective observer trying to provide comfort to an old companion.

Roderick is the last male heir of the ancient Usher family lineage. He suffers from an acute nervous condition that causes intense paranoia, hypersensitivity to sensory stimuli, physical weakness, and wildly fluctuating moods. Confined to the house for years, he spends his time painting, reading, and playing frantic guitar improvisations while dreading an inevitable doom related to his family's ancestral home.

Madeline is Roderick's twin sister and the only other living member of the Usher family. She suffers from a mysterious wasting disease characterized by physical apathy and sudden periods of bodily rigidity. She lives in total isolation within the mansion walls, interacting only with her brother and her attending doctors as her physical state steadily declines.

The family physician is the doctor tasked with diagnosing and treating Madeline's confusing wasting illness. He walks the dark halls of the Usher estate with a furtive, nervous energy, seemingly baffled by the nature of the family's afflictions. His behavior suggests he harbors ulterior motives or unspoken suspicions regarding his patients.

The valet is a silent servant working at the House of Usher. He waits at the entrance to receive guests and escorts the visiting narrator through the winding, poorly lit passages of the mansion to reach Roderick's private quarters.