Basic dyes such as malachite green and methylene blue have been used as disinfectants to control water fungal infections since the 1930s. However, after succeeding reports of carcinogenicity and bioaccumulation of the dye, their use was forbidden in lieu of public health. This study undertook to evaluate the therapeutic effect of sulfur solution processed by effective microorganisms (EM-PSS) against Saprolegnia parasitica infection, and its safety in fish. In vitro antifungal evaluation of EM-PSS inhibited the growth of S. parasitica mycelia at concentrations of 50 ppm or higher. The acute toxicity test of EM-PSS to the mud fish (Misgurnus mizolepis) measured a no effect concentration (NOEC) at 100 ppm, the lowest effect concentration (LOEC) at 125 ppm, and the half-lethal concentration (LC50) at 125 ppm in juvenile and 250 ppm in the immature stage. In addition, the ecotoxicity test of EM-PSS using Daphnia magna inhibited swimming of D. magna at concentrations of 100 ppm or less. Lastly, the EM-PSS prevented infection of S. parasitica to mud fish, at concentrations of 50 ppm. Furthermore, at 100 ppm concentration, the EM-PSS showed no acute toxicity on mud fish, nor any eco-toxic effects on D. magnano. Therefore, we conclude that carcinogenic disinfectants such as malachite green and methylene blue could be replaced by EM-PSS to remove S. parasitica in mud fish farming, and might be a potential eco-friendly disinfectant in aquaculture.