Like many of the tales Hawthorne wrote during his time living in The Old Manse, "The Birth-Mark" discusses the psychological impact in sexual relations. The birthmark does not become an issue to Aylmer until after the marriage, which he suddenly sees as sexual: "now vaguely portrayed, now lost, now stealing forth again, and glimmering to-and-fro with every pulse of emotion". Written shortly after Hawthorne married Sophia Peabody, the story emphasizes the husband's sexual guilt disguised as superficial cosmetology.
Aylmer's pursuit of perfection is both tragic and allegorical. The irony of Aylmer's obsession and pursuit is that he was a man whose "most splendid successes were almost invariably failures." Rather than obsessing over correcting his failures, he quickly forgets them. Similarly, instead of obsessing over Georgiana's splendid beauty, he quickly forgets it. That a man of so many failures would be trying to perfect someone else is both ironic and allegorical. This type of story has biblical symmetry to Jesus's "Sermon on the Mount." In Matthew 7:3, Christ is quoted as saying, "Why do you see the speck that is in your brother's eye, but do not notice the log that is in your own eye?" Aylmer's unyielding pursuit to remove the one "flaw" from Georgiana shows his own blindness of conscience. Georgiana's death is foreshadowed in Aylmer's dream of cutting out the mark, in which he discovers the birthmark is connected to her heart. He elects to cut out her heart as well in his attempt to remove the birthmark.
Other critics, like Stephen Youra, suggest that, to Aylmer, the birthmark represents the flaws within the human race—which includes "original sin", which "woman has cast men into"—and because of this, elects it as the symbol of his wife's "liability to sin, sorrow, decay, and death". Others suggest we view the story "as a story of failure rather than as the success story it really is — the demonstration of how to murder your wife and get away with it".
Hawthorne may have been criticizing the epoch of reform in which he was living, and specifically calling attempts at reform ineffective and the reformers dangerous.  The story is often compared to Edgar Allan Poe's "The Oval Portrait".
Aylmer is a scientist and husband to Georgiana. Robert B. Heilman suggests that Aylmer has taken science as his religion and that Aylmer’s views on "the best that the Earth could offer" is "inadequate". Heilman further says that "the mistake Aylmer makes" is the "critical problem" with the story, in that he has "apotheosized science".
Georgiana is the wife of Aylmer and, as Sarah Bird Wright puts it, the "doomed heroine" of the story. Georgiana agrees to allow Aylmer to experiment on her in an attempt to remove her birthmark—which turns out to be a fatal decision. Wright quotes Millicent Bell's thoughts on Georgiana's final words by saying they are "indicative of Hawthorne’s struggle with romanticism... he yearns to depict life as found".
Aminadab, Aylmer’s laboratory assistant, is described as being short and bulky with a shaggy appearance; Aylmer addresses him as "thou human machine" and "thou man of clay." Wright refers to Nancy Bunge's observation that "because Aminadab possesses vast physical strength and 'earthiness' he undertakes to perform unpleasant tasks in order to free Aylmer to 'cultivate delusions of transcendence'". Judith Fetterley suggests that "Aminadab symbolizes the earthly, physical, erotic self that has been split apart from Aylmer". Critic Evan Lang Pandya contends that Aminadab, whose characterization hearkens after that of the Greek god Hephaestus, represents pagan elements in Puritan New England culture.