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Saprolegnia infection of freshwater fish causes considerable economic loss to the aquaculture industry. Acidic electrolytic water (AEW) is highly valued by researchers for its rapid, efficient and broad-spectrum advantages in disinfection. This study investigated the bactericidal effect, mechanism and application of AEW on Saprolegnia. The results showed that AEW had a bactericidal effect on both Saprolegnia mycelia and spores, yet the bactericidal effect on Saprolegnia mycelia were superior. The available chlorine concentration (ACC), oxidation-reduction potential (ORP) and treatment time were the main factors affecting the bactericidal effect of AEW. AEW mainly affected the normal physiological function of mycelium by destroying the cell membrane structure and causing leakage contents. However, the mycelium treated with AEW showed a decrease in antioxidant enzyme activity, which led to a massive outbreak of reactive oxygen species (ROS) and eventually induced apoptosis and necrosis. To investigate the safety assessment of AEW for grass carp and its application in Saprolegnia disease control, the results showed that AEW with dilution greater than 100 is safe for grass carp juveniles and has a preventive effect on Saprolegnia infection in grass carp. In conclusion, AEW provides a new technical method for the prevention and control of Saprolegnia in the future, and it has potential application in the aquaculture industry.