The film consists of a series of tightly interlinked vignettes, the most sustained of which details the story of a man and a woman who are passionately in love. Their attempts to consummate their passion are constantly thwarted, by their families, by the Church and bourgeois society in general.

Mathilde Strangerson, the daughter of an eminent scientist, narrowly escapes being murdered in her own bedroom by an unknown assailant. As the room was locked from the inside, no one can understand how the attacker managed to enter or leave the room. Reporter and amateur sleuth Joseph Rouletabille arrives on the scene to protect Mathilde and resolve the mystery of the yellow room.