Aside from a family doctor, servant, and valet, who briefly appear in the beginning of the story, the characters of "The Fall of the House of Usher" include the narrator, Roderick Usher, and Madeline Usher.

Though the plot primarily focuses on the central character of Roderick, a typical Poe character, much of the story's tone and mystery come from Madeline and the narrator: Madeline's presence pervades the story, contributing to its tense atmosphere, and the narrator's observations and conclusions establish the short story's mystery and major themes.

The unnamed narrator is the first character introduced, and he tells the story from his perspective. He is a childhood friend of Roderick’s and says little else about himself except to give his impressions of the story's events. Throughout “The Fall of the House of Usher” he notices strange things, such as the mist rising from the tarn, Roderick’s growing madness, and his own feelings of fear, but he often dismisses them as products of his imagination or superstition. He attempts to explain any strange event with reason, approaching the situation rationally. He refers to Roderick as a hypochondriac and believes that his strange illness is illusory. However, at the end of the story, the narrator never states definitively whether or not he believes the events at the House of Usher are supernatural or merely the product of fear and the mind. He is the only witness to the end of the Usher line. He is the only one to see the Ushers die and the house sink into the tarn. He leaves the House of Usher in a state of panic and ends his story there.

Roderick Usher and the narrator are childhood friends, but Roderick has changed drastically since the narrator last saw him. He is now pale and wispy-haired. In the beginning, he goes back and forth from sullenness and nervous agitation to liveliness. A mysterious and incurable illness plagues him and causes his senses to be highly reactive. It is unclear whether he is actually sick or if he is a hypochondriac who is going insane from fear and isolation.

Roderick spends his time reading, writing music, and painting. The painting that Roderick creates is of a long, dark, underground tunnel. Strange beams of light glow from the canvas, and the narrator finds them grotesque. Roderick also writes a song about a prosperous palace that falls victim to evil and sorrow. The song and the painting reflect Roderick’s feelings about the decay of his home, the inevitable end of his family line, and his own declining health.

When Madeline dies, Roderick has her buried quickly in a basement vault. Roderick’s condition deteriorates, and he abandons his former hobbies, taking to hurriedly roaming through the house and staring into vacant corners. He is superstitious and constantly afraid. The night of the storm, Roderick comes to the narrator’s room in a state of hysteria and shows him the glowing atmosphere emanating from the house and the grounds. When the narrator begins to read a story to calm him, Roderick sits in a chair and mumbles. In his mumblings he reveals that he has been hearing scrapings from Madeline’s vault all week, and he fears he buried her alive. The prospect of burying Madeline prematurely fills him with guilt, contributing to his descent into madness. All of Roderick’s worst fears are confirmed when Madeline returns and collapses on him, killing them both. The house sinks into the tarn, forever burying the last of the Usher line.

Madeline only appears in three scenes in the story: once roaming through the house, once when she is interred, and later when she returns from a premature burial. She is Roderick’s twin and described as young and beautiful. However, she suffers from an unknown disease the doctors cannot cure. They describe her illness as “a settled apathy, a gradual wasting away of the person.” Roderick prematurely buries her after her supposed death, but both the narrator and Roderick hear her attempts to escape without comprehension. She reappears on the night of a terrible storm only to collapse on her brother and die, inadvertently killing Roderick as well. Her death and the narrator’s flight from the house are the final acts before the house sinks.