Abstract: To evaluate the inhibitory effect and application potential of Epicoccum nigrum Z01 fermentation broth against Saprolegnia, this study isolated a filamentous fungus from the eyes of grass carp (Ctenopharyngodon idella) and obtained an antagonistic fungus against Saprolegnia from culture plates. After morphological and molecular biological identification, the strains were confirmed as Saprolegnia ferax and Epicoccum nigrum Z01, respectively. The inhibitory effect of the fermentation broth on S. ferax was assessed using plate confrontation, agar dilution, and broth dilution assays. Safety was evaluated through histopathological observation, hematological parameter analysis, and survival rate statistics after fish immersion. Furthermore, the preventive efficacy of the fermentation broth was assessed in grass carp infected with Saprolegnia via injection, immersion, and oral administration, while its therapeutic effect was investigated through post-infection immersion. Results demonstrated that E. nigrum Z01 exerted a significant and stable inhibitory effect on Saprolegnia growth. In the agar dilution assay, no Saprolegnia growth occurred when the fermentation broth-to-PDA ratio exceeded 3:7, and no spore germination was observed in PDB supplemented with more than 0.1 mL of fermentation broth. Immersed fish exhibited normal blood cell morphology and higher survival rates than the control group. The prevalence of S. ferax infections in grass carp treated by immersion, injection, and oral administration was significantly reduced by 60%, 50%, and 40%, respectively. Importantly, E. nigrum Z01 markedly suppressed Saprolegnia growth without causing damage to the liver, spleen, trunk kidney, and 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.