Anne is an imaginative, talkative 11-year-old girl living in Amsterdam after her family fled Germany. She attends a Montessori school where her creativity thrives, though her constant chattering sometimes frustrates her mother. She loves mythology, fairy tales, and reading, and she pays close attention to the natural world, particularly the magpies and rabbits in her neighborhood. As the Nazi occupation takes hold and curtails her freedoms, she begins writing down her observations, using stories to make sense of the growing danger around her.

Margot is Anne's older sister, turning 15 and 16 during the early years of the occupation. She is a dedicated student who tries to supervise Anne's energetic behavior on their walks home. Her mother views her as obedient and responsible. Under the strain of the occupation, Margot relies on lists and daily schedules to maintain order, harboring a specific goal to become a nurse or midwife in Jerusalem.

Otto is a businessman who runs a jam and spice company in Amsterdam. He moved his family from Germany to the neutral Netherlands to escape the Nazi regime. He attempts to maintain a calm exterior and relies on legal channels, applying repeatedly for travel visas to the United States. He possesses a deep appreciation for literature, taking time to share Greek myths and Charles Dickens novels with Anne.

Edith is the mother of Anne and Margot. She presents a pragmatic, no-nonsense attitude and expects good behavior, causing friction with her more spirited younger daughter. Beneath her strict exterior, she harbors intense anxiety about her family's safety under the occupation. She hides family heirlooms under the sink and quietly manages the household's diminishing resources.

Rosa is Edith's mother, who joined the Franks in Amsterdam after surviving Kristallnacht in Germany. She shares a special kinship with Anne, encouraging her granddaughter to speak freely and live fully. She suffers from a severe illness but keeps her physical pain and her fears about the Nazi threat hidden to protect her family's spirits.

Miep is a smart, self-assured Austrian woman who works in Otto Frank's office. She faces danger of deportation because of her Austrian citizenship and her refusal to join a Nazi women's group. She marries her Dutch fiancé, Jan, to secure her citizenship and maintains a steadfast devotion to helping the Frank family.

Helmut is a tall, handsome teenager sent to Amsterdam by his parents in Germany for safety. He dreams of emigrating to America and teaches himself English in preparation. He falls intensely for Anne and plots ways they might escape the occupation together, displaying an urgent need to experience life before time runs out.

Nathan is an American businessman who knew Otto when Otto lived in New York as a young man. He represents the Franks' hope for escape, attempting to assist Otto with immigration papers when the family seeks to leave the Netherlands.

Hermann is a business associate of Otto Frank. He collaborates with Otto to secretly stock the upper floors of an annex building with food and supplies, preparing a refuge for both of their families.

Jan is a Dutch citizen whose marriage to Miep provides her with legal protection in Amsterdam. He joins Otto's company board to help protect the business from Nazi seizure and assists in transporting supplies for the Franks.

Hanneli is one of Anne's close friends in Amsterdam. When Jewish students are expelled from regular schools, she transfers to the same Jewish-only classroom that Anne is forced to attend.

Sanne is a childhood friend of Anne's. She visits the Frank apartment to play games and read books before the occupation severely restricts their activities and movements.

Jacque is a new friend Anne makes after being forced out of her Montessori school. She invites Anne to spend the night at her apartment during the difficult winter months.

Old Edgar is a Sephardic Jewish book dealer whose ancestors fled the Inquisition. He secretly continues his trade after the Nazis ban Jewish people from selling books.

Clara is Old Edgar's wife. She interacts with Anne and Otto when they visit the market, specifically encouraging Anne to purchase a book of Greek and Roman myths instead of a traditional novel.