Atovaquone, a substituted hydroxynaphthoquinone, is a potent antimalarial drug that acts by inhibiting the parasite’s mitochondrial cytochrome bc₁ complex (cyt bc₁). Mutations in cyt bc₁ confer atovaquone resistance. Here we describe the X-ray structure of mitochondrial cyt bc₁ from Saccharomyces cerevisiae with atovaquone bound in the catalytic Qo site, at 3.0-Å resolution. A polarized H-bond to His181 of the Rieske protein in cyt bc₁ traps the ionized hydroxyl group of the drug. Side chains of highly conserved cytochrome b residues establish multiple non-polar interactions with the napththoquinone group, whereas less-conserved residues are in contact with atovaquone’s cyclohexyl–chlorophenyl tail. Our structural analysis reveals the molecular basis of atovaquone’s broad target spectrum, species-specific efficacies and acquired resistances, and may aid drug development to control the spread of resistant parasites.