Giovanni is a young student from southern Italy who comes to the University of Padua to continue his studies. Lacking funds, he takes up residence in an old, gloomy building once owned by a noble family. He is highly conscious of social expectations and quickly develops a fascination with the mysterious young woman residing in the neighboring garden.

Beatrice is a vibrant, extraordinarily beautiful young woman who lives a sheltered life within her father's walls. She assists him by tending to a magnificent but unusual garden, showing a particular affection for a striking purple plant located by a broken fountain. Despite her profound isolation from the outside world, she remains selfless and vivacious.

Doctor Rappaccini is a tall, emaciated, and sickly-looking older man dressed in black scholar's garb. As a famous scientist in Padua, he is reputed to derive remarkable medicines from toxic plants. His intense dedication to his botanical experiments often makes him appear cold and indifferent to ordinary human concerns.

Professor Baglioni is an elderly, jovial professor of medicine at the University of Padua. As an old friend of Giovanni's father, he takes it upon himself to look out for the young student. He holds traditional views on medicine and harbors a deep-seated distrust toward the experimental methods of his academic peers.

Lisabetta is the landlady of the ancient, dilapidated aristocratic house where Giovanni takes up residence. She takes a quick liking to the young student and serves as a facilitator of his early curiosity about the neighboring property.