Epsilon-polylysine (ε-PL) is a natural antimicrobial and has been widely used as a food preservative. Previous studies showed ε-PL being effective for control of foodborne bacteria; however, little is known about its control effects on postharvest pathogenic fungi and the possible modes of action. Here, we found that ε-PL could significantly inhibit mycelial growth, spore germination and germ tube elongation of Botrytis cinerea in vitro, as well as effectively control gray mold in harvested jujube fruit. Further investigation indicates that the modes of action of ε-PL against B. cinerea include (i) stimulating the accumulation of intracellular reactive oxygen species (ROS) and reducing the expression of pathogenesis-related genes of the pathogen, (ii) resulting in the leakage of soluble carbohydrates and nucleic acids, as well as the damage of plasma membrane integrity of the fungal cells, (iii) inducing the expression of respiratory burst oxidase homolog (RBOH) genes in ε-PL-treated jujube fruit.