As a natural antimicrobial food additive, εpsilon-poly-lysine (ε-PL) was reported to be effective in preventing food spoilage. However, few studies regarding the inhibitory effect of ε-PL against phytopathogenic fungi, especially post-harvest pathogens, were reported. In this study, the antifungal activity of ε-PL via membrane damage in Penicillium digitatum was investigated. The data showed that mycelial growth, spore germination rate, and germ tube length of P. digitatum were markedly inhibited by ε-PL. Moreover, ε-PL was also effective in reducing the lesion diameter in citrus fruit. Scanning electron microscopy showed that mycelial morphology was seriously damaged after ε-PL treatment. Electrical conductivity measurement and propidium iodide assays showed that P. digitatum lost plasma membrane integrity by ε-PL. The level of malondialdehyde demonstrated that ε-PL led to lipid peroxidation in the fungal pathogen. These results indicate that ε-PL may serve as a sustainable partial substitute for chemical pesticides.