Abstract: Saprolegnia is extremely harmful in freshwater fish farming and long-distance transport, often leading to the death of infected fish. In this study, a filamentous fungus was isolated from the eyes of grass carp (Ctenopharyngodon idella), and an antagonistic fungus against Saprolegnia was obtained from culture plates. Morphological and molecular biological identification confirmed these strains as Saprolegnia ferax and Epicoccum nigrum Z01, respectively. The inhibitory effect of E. nigrum Z01 fermentation broth on Saprolegnia was evaluated using the plate confrontation, agar dilution, and broth dilution assays. Safety assessments included histopathological observation, hematological parameter analysis, and survival rate statistics after fish immersion. Furthermore, the preventive efficacy of the fermentation broth was assessed through injection, immersion, and oral administration in Saprolegnia-infected grass carp, while its therapeutic effect was investigated via post-infection immersion. Results demonstrated that E. nigrum Z01 exerted a significant and stable inhibitory effect on Saprolegnia growth. In the agar dilution method, no Saprolegnia growth occurred when the fermentation broth-to-PDA ratio exceeded 3:7, and no spore germination was observed in PDB supplemented with 0.1 mL of fermentation broth. Immersed fish exhibited normal blood cell morphology and higher survival rates than the control group. Significant reductions of 60%, 50%, and 40% in the prevalence of S. ferax infections in grass carp treated by immersion, injection and gavage treatment. Importantly, E. nigrum Z01 markedly suppressed Saprolegnia growth without causing damage to the liver, spleen, kidney, or gut of grass carp. The fermentation broth also reduced infection rates while promoting choroid thickening and increasing lamina propria cell counts in intestinal villi. These findings provide crucial insights for the biological control of Saprolegnia.