Methyl thujate, a monoterpenoid substance, was effective in controlling postharvest gray mold caused by Botrytis cinerea on apple fruit, during application in aqueous solution. Methyl thujate strongly inhibited spore germination, germ tube elongation and mycelial spreading of B. cinerea in the culture medium, and the inhibition on fruit decay was closely correlated with methyl thujate concentrations. By means of propidium iodide and fluorescein diacetate staining, loss of membrane integrity and cell viability in B. cinerea was detected after methyl thujate treatment. Furthermore, methyl thujate led to cytoplasmic accumulation of reactive oxygen species, leakage of cellular constituents and variations in cell wall component deposition in B. cinerea hyphae, as the genes encoded subunits for NADPH oxidase were synergistically upregulated, whereas the genes involved in cell wall modeling and virulence responded differentially. Taken together, these data suggest that the mechanisms by which methyl thujate decreased fruit decay may be directly attributed to the induced ROS burst, breakdown of cell membrane integrity and loss of cytoplasmic contents from the hyphae.