Microbial secondary metabolites produced by are applied to control plant diseases. The metabolite, ε-poly--lysine (ε-PL), is a non-toxic food preservative, but the potential application of this compound as a microbial fungicide in agriculture is rarely reported. In this study, the effect and mode of action of ε-PL on two necrotrophic pathogenic fungi, and , were investigated. The results showed that ε-PL effectively inhibited the mycelial growth of and with EC values of 283 μg/mL and 281 μg/mL, respectively. In addition, ε-PL at the dose of 150 and 300 μg/mL reduced sclerotia formation. The results of the RNA-seq and RT-qPCR validation indicated that ε-PL significantly regulated the gene expression of critical differential expressed genes (DEGs) involved in fungal growth, metabolism, pathogenicity, and induced an increase in the expression of the fungal stress responses and the detoxification genes. These results provided new insights for understanding the modes of action of ε-PL on and and improved the sustainable management of these plant diseases.