Plant-derived essential oils (EOs) offer a promising alternative as potential natural antimicrobial agents. However, the unclear mechanism of their components limits their precise application. Nerolidol, a sesquiterpene alcohol abundant in EOs of various plants, has known antifungal activities, but its mechanism against Botrytis cinerea is unclear. In this study, the inhibitory activity of nerolidol on B. cinerea and the underlying mechanism were investigated. Nerolidol inhibited growth and spore germination of B. cinerea, and effectively controlled grey mold in strawberry and tomato fruit. Transcriptomic analysis revealed that nerolidol reprogrammed the metabolic pathways of B. cinerea, leading to changes in many important metabolism-related genes. Specifically, nerolidol caused disruption of peroxisome structure, downregulation of hydrogen peroxide (H₂O₂) and lipid metabolism, and accumulation of reactive oxygen species (ROS). The ROS scavenger N-acetylcysteine can partially reverse the growth inhibition of B. cinerea and alleviate peroxisome damage. Our results suggest that peroxisome disruption and H₂O₂ accumulation contribute to the antifungal activity of nerolidol against B. cinerea.