Sclerotinia sclerotiorum, a pathogenic fungus of oilseed rape, poses a severe threat to the oilseed rapeseed industry. In this study, we evaluated the potential of the natural compound hinokitiol against S. sclerotiorum by determining its biological activity and physiological characteristics. Our results showed that hinokitiol strongly inhibited the hyphae expansion of S. sclerotiorum, and its effective concentration of hyphae growing inhibition by 50% (EC50) against 103 S. sclerotiorum strains varied from 0.36 to 3.45 μg/mL, with an average of 1.23 μg/mL. Hinokitiol possessed better protective efficacy than therapeutic effects, and it exhibited no cross-resistance between carbendazim. After treatment with hinokitiol, many vesicular protrusions developed on the mycelium with rough surface and thickened cell wall. Moreover, the cell membrane permeability and glycerol content increased, while the oxalic acid declined after hinokitiol treatment. In addition, hinokitiol induced membrane lipid peroxidation and improved the production of reactive oxygen species (ROS) in S. sclerotiorum. Importantly, real-time quantitative polymerase chain reaction showed that cell wall and ROS synthesis-related genes were significantly up-regulated after hinokitiol treatment. This study revealed that hinokitiol has good biological activity against S. sclerotiorum and could be considered as an alternative bio-fungicide for the resistance management in controlling sclerotinia stem rot infected by S. sclerotiorum. These investigations provided new insights into understanding the toxic action of hinokitiol against pathogenic fungi. © 2024 Society of Chemical Industry.