Zoe is the titular octoroon, meaning she is of one-eighth Black heritage. She is the illegitimate daughter of the late Judge Peyton and lives at the Terrebonne plantation. Though she was raised with great affection and believed to be a free woman, the judge's outstanding debts complicate her legal standing, threatening to return her to slavery. She possesses a kind heart and is highly educated, making her beloved by nearly everyone on the estate.

George is Mrs. Peyton's nephew and the newly arrived heir to the heavily indebted Terrebonne estate. Having spent significant time in Europe, he returns to the South and quickly develops feelings for Zoe. He dismisses the strict racial divisions of his society, expressing a willingness to overlook the laws prohibiting their romance in favor of his genuine affection.

Jacob McCloskey is a Northern Yankee who formerly served as an overseer at Terrebonne and now holds a major mortgage on the property. Feeling inadequate and resented among the wealthy Southern planters, he schemes to take full control of the estate. He harbors an intense, unreciprocated obsession with Zoe and actively works against the Peyton family's interests.

Salem Scudder is a Yankee from Massachusetts who serves as an overseer at Terrebonne. A man of many past trades, including photography and inventing, he owns half the estate but feels deep remorse for his role in the plantation's financial decline. He maintains a helpful demeanor and tries to assist Mrs. Peyton, setting him apart from McCloskey.

Mrs. Peyton is the widow of the late Judge Peyton and the matriarch of Terrebonne. She faces the impending loss of her home and the people she enslaves due to her late husband's severe financial debts. She is a hospitable woman who cares deeply for Zoe and views the enslaved people on her estate with maternal affection.

Old Pete is a seventy-two-year-old enslaved man at Terrebonne who is considered too old to perform hard labor. He acts as a subservient, lighthearted figure on the estate, demonstrating deep loyalty to the Peyton family and serving to keep the spirits of the other enslaved workers high.

Dora is a wealthy Southern belle whose substantial fortune represents a potential financial salvation for the indebted Terrebonne estate. Accustomed to a life of extreme privilege, she hopes George Peyton will court her, occasionally showing a dismissive attitude toward Zoe and the enslaved workers.

Mr. Sunnyside is a wealthy neighboring plantation owner and Dora's father. He acts as a hospitable and supportive presence to Mrs. Peyton, offering advice and assistance as she attempts to manage her massive debts.

Paul is a thirteen-year-old enslaved boy at Terrebonne who is one-quarter Black. Because he was a favorite of the late Judge Peyton, he is largely spared from difficult labor and is well-liked by the residents. He shares a close, devoted friendship with Wahnotee.

Wahnotee is a Native American chief from the Lepan tribe who stays near the Terrebonne estate. Despite facing heavy discrimination and stereotyping from the white characters, he possesses a gentle demeanor and remains on the plantation entirely out of his deep, protective affection for the young boy Paul.

Dido is an enslaved woman who works as the cook at the Terrebonne estate. She possesses knowledge of medicinal liquids and remedies, which causes Zoe to seek her out during a moment of distress.

Captain Ratts is the commander of the Steamer Magnolia. He participates in the local events surrounding the Terrebonne estate and interacts with the plantation owners regarding the transport of goods and people.

Grace is an enslaved woman at the Terrebonne estate with young children. Her future on the plantation is thrown into uncertainty when the estate faces foreclosure and the residents are forced to auction their property.

Minnie is an enslaved woman at Terrebonne tasked with attending to the visiting Dora Sunnyside, highlighting the clear class and racial divides of the setting.

Solon is an enslaved man at Terrebonne who relays important information to the plantation overseers and owners during times of high tension.

Colonel Pointdexter is a local figure and participant in the estate's affairs. He represents the strict adherence to Southern laws, insisting on formal legal processes regardless of his personal feelings toward the people involved.