Objective:The aim of this study is to evaluate the antimicrobial efficacy of 2.5% sodium hypochlorite, 2% chlorhexidine, and ozonated water on biofilms of Enterococcus faecalis, Streptococcus mutans, and Candida albicans in mesiobuccal root canals with severe curvature of mandibular molars.Materials and Methods:This was an experimental ex vivo study in microbiologic laboratory. Sixty mesiobuccal root canals with severe curvature of mandibular molars were contaminated with standard strains of E. faecalis, S. mutans, and C. albicans. The specimens were randomly divided into four groups (n = 15) according to irrigating solution: SH: 2.5% sodium hypochlorite; CH: 2% chlorhexidine; O3: ozonated water; and control: double-distilled water. The mesiobuccal root canals of all groups were instrumented with the WaveOne Gold Primary reciprocating system. Three cycles of instrumentation with three short in-and-out brushing motions were performed: (1) in the coronal third, (2) in the middle third, and (3) in the apical third of the canal. A ProGlider file was used before the first cycle.Statistical Analysis:Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple comparison test. Samples were collected for viable bacterial counts before and after instrumentation.Results:All groups showed significant biofilm reduction after irrigation (P < 0.01). After instrumentation, sodium hypochlorite (98.07%), chlorhexidine (98.31%), and ozonated water (98.02%) produced a significantly reduction in bacterial counts compared with double-distilled water (control, 72.98%) (P < 0.01).Conclusion:All irrigants tested in this study showed similar antimicrobial activity. Thus, ozonated water may be an option for microbial reduction in the root canal system.