Objective In this study, we tested different disinfectants and different action times to establish an efficient protocol for zebrafish embryo disinfection, which may in turn improve the management of zebrafish facilities. Methods We tested three reagents that can be used for embryo disinfection and are readily available in the domestic market: reagent-grade povidone-iodine, sodium hypochlorite, and chlorine dioxide. We evaluated the toxic effects of the reagents at three concentrations and two action times on zebrafish embryos at three stages, in term of survival, dechorioning, and malformation rates. The effects of the three disinfectants were also compared in terms of the amounts of embryos with surface bacteria after disinfection. Results Chlorine-containing disinfectants, i. e. sodium hypochlorite and chlorine dioxide, were better able to kill bacteria on the embryo surface, while povidoneiodine was not quite effective. Survival, dechorioning, and malformation rates were similar in embryos treated with 30 ppm sodium hypochlorite or chlorine dioxide to those in control embryos. Treatment with chlorine-containing disinfectants for 10 min were more effective in sterilizing than for 5 min. Conclusions The result of this study suggest treating 6 ~ 30 hpf embryos with 30 ppm sodium hypochlorite for 10 min as an operational method.