Abstract: To investigate the effect of coumarin on Botrytis cinerea, the antifungal activity, enzyme activity and conductivity were determined. Then the antifungal activity, protein content, enzyme activity and cell membrane permeability were analyzed to reveal the mechanism of coumarin to B. cinerea. The results showed that the EC₅₀ value of coumarin to B. cinerea on liquid medium (PD medium) was 101 mg/L. The protein concentration of the treatment group was lower than that of the control either intracellularly or extracellularly. When the concentration of coumarin was 101 mg/L, the inhibition rates of coumarin on adenosine triphosphatase (ATPase) and catalase (CAT) in B. cinerea were 62.60% and 76.50%, respectively. And those on cellulase (CL), polygalacturonase (PG) and pectinase were 57.63%, 64.51% and 61.13%, respectively. Moreover, coumarin treatment significantly enhanced cell membrane permeability. After the treatment, the conductivity became 17568.79 μS/cm at 48 h, which was 1.9 times of that of the control group. Therefore, results demonstrated that coumarin can inhibit the growth of B. cinerea, hinder its respiratory metabolism, interfere with the permeation of the cell membrane, and inhibit enzyme activity, which may provide theoretical support for the prevention of gray mold with coumarin, a new plant-derived fungicide.