Objective To conduct an epidemiological investigation of several pathogens in zebrafish, and to provide the basis for subsequent development of relevant standards.Methods This study mainly used traditional methods based on morphological, physiological and biochemical characteristics of bacteria, combined with molecular biology methods, to isolate bacteria and artificially infect healthy zebrafish with the bacteria. Through investigating the incidence and mortality in the experimental group, we distinguished the virulent effect of the pathogens, and further understanding the pathogens affecting the health of zebrafish.Results There were three kinds of pathogens identified in all sampling institutions, including pathogenic Aeromonas hydrophila, Aeromonas sobria and Pseudomonas aeruginosa. Aeromonas hydrophila was detected in most sampling institutions, and showed to possess strong virulence. Aeromonas sobria and Pseudomonas aeruginosa were detected in some sampling institutions, and the virulence of Aeromonas sobria was revealed to be weaker than that of Aeromonas hydrophila. The virulence of Pseudomonas aeruginosa was the weakest among these three bacteria, did not induce typical clinical symptoms and death in the zebrafish.Conclusions Aeromonas hydrophila may cause contamination of fish experimental facilities, and it has been shown to be potentially able to infect humans. Thus regarding the fish infection routes, enough attention should be payed to Aeromonas hydrophila, and it should be the focus in the pathogens monitoring.