Water serves as a critical vector for the transmission of pathogenic microorganisms, playing a pivotal role in the emergence and propagation of numerous diseases. Ozone (O3) disinfection technology offers promising potential for mitigating the spread of these pathogens in aquatic environments. However, previous studies have only focused on the inactivated effect of O3 on a single pathogenic microorganism, lacking a comprehensive comparative analysis of various influencing factors and different types of pathogens, while the cost-effectiveness of O3 technology has not been mentioned. This review synthesized the migration characteristics of various pathogenic microorganisms in water bodies and examined the properties, mechanisms, and influencing factors of O3 inactivation. It evaluated the efficacy of O3 against diverse pathogens, namely bacteria, viruses, protozoa, and fungi, and provided a comparative analysis of their sensitivities to O3. The formation and impact of harmful disinfection by-products (DBPs) during the O3 inactivation process were assessed, alongside an analysis of the cost-effectiveness of this method. Additionally, potential synergistic treatment processes involving O3 were proposed. Based on these findings, recommendations were made for optimizing the utilization of O3 in water inactivation in order to formulate better inactivation strategies in the post-pandemic eras.