Edwardsiella tarda (E. tarda) and Vibrio harveyi (V. harveyi), common pathogenic bacteria in fish, contribute to elevated mortality rates within fish populations. Chlorination, a widely utilized disinfection method, can effectively manage these microorganisms. The present research examined the impact of chlorination in the inactivation of two microorganisms, with varying pH, chlorine doses, natural organic matter (NOM) concentrations, and temperatures. Microbial inactivation during seawater chlorination is strongly influenced by the formation of chlorine-produced oxidants (CPOs), whose speciation was determined by pH, and concentrations of bromide ions and ammonium ions. At pH 7.1, mixed CPOs (mainly HOBr/NHxBry/NHBrCl) were formed, while NH2Cl was the dominant species at pH 8.2. The higher inactivation efficacies at pH 7.1 compared with those at pH 8.2 were explained by the stronger bactericidal activities of HOBr than NH2Cl. Meanwhile, microbial inactivation was enhanced with increasing chlorine dose and temperature, but it was inhibited with increasing NOM concentration. For a 2 log reduction of E. tarda and V. harveyi, the required concentration–time product (Ct) values were 0.0978 and 0.1102 mg·min/L, respectively, at pH 7.1. These values rose to 0.3644 and 0.3968 mg·min/L, respectively, when the pH increased to 8.2. The Ct values determined in this study provide essential guidelines for safeguarding against microbial contaminants in seawater, thus proposing effective chlorination protocols in aquaculture.