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The widespread use of synthetic fungicides has gradually increased drug resistance and has negatively impacted human health and caused environmental contamination. This study aimed to investigate the effects and mechanisms of action of protocatechuic acid methyl ester (PCAME), a plant-derived biologically active compound, against decay mold disease of postharvest strawberry fruit caused by Botrytis cinerea. PCAME inhibited in vitro mycelial growth and spore germination of B. cinerea and decreased the susceptibility of strawberries to B. cinerea invasion, as revealed by the lesion area. PCAME effectively altered the transcriptional profile of B. cinerea and enhanced the transcription of genes related to oxidative stress and cell membrane lipid peroxidation, ultimately causing membrane disturbance and cell death. These results were verified by increased extracellular conductivity, leakage of cytoplasmic constituents including sugars and nucleotides, and propidium iodide staining. Furthermore, PCAME treatment activated the expression of genes involved in antioxidant metabolism and defense-related proteins in strawberries infected with B. cinerea. These data suggest that PCAME inhibits postharvest gray mold disease in strawberries through direct antifungal activity and the enhancement of host resistance responses. PCAME can be considered as natural agent that alleviates postharvest gray mold decay in strawberries. [Display omitted] • PCAME exhibited in vitro and in vivo antifungal activity against B. cinerea. • PCAME disturbed the integrity of cell membrane of B. cinerea. • PCAME caused oxidative stress and membrane lipid peroxidation of B. cinerea. • PCAME induced resistance responses of strawberries. [ABSTRACT FROM AUTHOR]