As Fifth Business is Dunstan Ramsay's autobiography, he serves as the central figure. The other characters are primarily presented through their interactions with him and their influence on his life. They are not depicted in a traditional novelistic manner. The most fully developed character is Boy Staunton, whom Ramsay refers to as his "lifelong friend and enemy" in the novel's second sentence. Staunton epitomizes worldly success. His early investment advice has ensured Ramsay's financial stability, allowing him to build a significant nest egg over the years. There is an element of romanticism in Boy Staunton. Following World War I, he idolizes the Prince of Wales: "Flaming Youth, and yet, withal, a Prince, remote and fated for great things. Just the very model for Boy Staunton, who saw himself in similar terms." For Staunton, surpassing his rivals in wealth and appearance is paramount. He confides in Ramsay, giving Ramsay insight into his social and domestic life. At home, Staunton fosters a dysfunctional family. He views his wife Leola as a failure, unfit for her social role, and attempts to reshape her. He excessively praises his daughter Caroline for her beauty and watches his son David closely for any signs of unmanliness. In his professional life, Staunton mentors several "clean-cut" young executives, whose careers decline after they marry and introduce their wives to Staunton. Ramsay observes that sex plays "a dominating part" in Staunton's life, though Staunton himself is oblivious, dismissing Freud as "a madman" for his emphasis on sex. After King Edward's abdication, Staunton's idol, Boy falls into a depression and temporarily leaves his family, driving Leola to a suicidal breakdown when Ramsay rebuffs her advances.

Following World War II and Leola's death, Boy Staunton ventures into public life, believing he can address Canada's issues by applying business strategies to governance. However, he struggles with the electorate due to his disdain for the common man and his overly handsome appearance for a politician. Denyse Hornick, recognizing his potential, manipulates him into marrying her and running for the largely ceremonial and expensive role of Lieutenant-Governor of Ontario. As he nears this goal, Boy dies under mysterious circumstances, found in a submerged car in Toronto harbor with a stone in his mouth. His death triggers Dunstan Ramsay's heart attack after he hears himself implicated by the Brazen Head in Magnus Eisengrim's magic show as one of Staunton's murderers, "the inevitable fifth, who was keeper of his conscience and keeper of the stone."

Mrs. Dempster's life is so deeply entwined with Ramsay's mythology that it becomes challenging to see her as an independent character. According to Ramsay, she believes she saves lost men's lives. Through three miracles, she revives Ramsay's brother, Willie, after he is presumed dead; watches over Ramsay during his coma after a battle injury; and transforms a tramp's life through an intimate encounter. This last act renders Mrs. Dempster infamous in Deptford. When discovered with the tramp in Deptford's gravel pit, she explains to her husband that she did it because the tramp "was very civil, 'Masa. And he wanted it so badly." This remark encapsulates Mrs. Dempster's unique nature. She is rational in her way but lacks understanding of societal norms and cannot mask her thoughts to fit them. Thus, she understands without comprehending: "She knew she was in disgrace with the world, but did not feel disgraced; she knew she was jeered at, but felt no humiliation. She lived by a light that arose from within." This inner light gives Mrs. Dempster a consistency that Ramsay interprets as saintliness, making her the laughing stock of Deptford and the target of ridicule from young boys.

Near the novel's conclusion, Mrs. Dempster becomes estranged from Ramsay after he reveals he has found Paul, her long-lost son. In her delusional mind, Paul remains a child rather than a man over forty. Ramsay, her sole connection to the outside world, now becomes her jailer, representing the forces keeping her son away. During his interaction with Mrs. Dempster, Ramsay uncharacteristically lies, as the truth would be unbearable. He would have to admit that Paul was not kidnapped but ran away because he could not handle the responsibility of her condition and the public scorn she attracted. Moreover, Paul has no desire to reunite with her. Consequently, Ramsay fabricates stories, and Mrs. Dempster perceives his deceit, identifying him as her adversary. This effectively ends their relationship, as Ramsay can no longer visit her.

Two other significant characters in the novel are Padre Ignacio Blazon, a Jesuit Bollandist, and Liselotte Vitzlipiitzli (Liesl), the manager of Eisengrim's touring magic show. Both serve as advisors to Ramsay. When it comes to Blazon, Ramsay seeks his guidance. Blazon views human existence as complex, filled with "marvels, cruel circumstances, obscenities, and commonplaces." He believes everything will be resolved with the coming of Christ, who will "declare the unity of the life of the flesh and the life of the spirit." Blazon advises Ramsay not to feel guilty about Mrs. Dempster's condition, suggesting that the real issue is "what figure" she represents in his "personal mythology." He explains that Mrs. Dempster's role in Ramsay's life is as much about him as it is about her, and that Ramsay must find his "answer in psychological truth, not in objective truth . . . And while you are searching, get on with your own life and accept the possibility that it may be purchased at the price of hers and that this may be God's plan for you and her."

Liesl is described as "the ugliest human creature" Ramsay has ever encountered. She dresses like a man, and Ramsay is profoundly disturbed to see her making love to the beautiful Faustina, a performer in Eisengrim's show. As Ramsay becomes more involved in critiquing and revising Eisengrim's magic show, he begins to talk openly with Liesl about himself. In turn, Liesl, who knows his personal history in detail, advises him to stop punishing himself by observing the excesses of others like Mrs. Dempster and the Stauntons, and to stop denying himself the pleasures of life. She identifies him as Fifth Business, a male character in an opera who "is the odd man out, the person who has no opposite of the other sex." Ramsay sees her as the devil—not a terrifying tempter, but his equal, someone who tells him things he needs to hear. One night, Liesl comes to his room and tries to make love to him, but Ramsay has no desire to be "ravished by a Swiss gargoyle," leading to a fight where Ramsay twists her nose so hard he thinks he "heard something crack." He emerges victorious in this encounter with the devil; after demonstrating his strength, he later makes love to Liesl following a long, civilized conversation as equals.