Aim: This study evaluated the disinfection ability of ozonated water in comparison to sodium hypochlorite (NaOCl) solution in root canals infected with Enterococcus faecalis. Material and methods: Forty single-rooted bovine teeth were standardized and prepared using K-files. The root canals were contaminated for 21 days and the first microbial sample was collected from root canals (S1). The teeth were divided into 5 groups: Saline solution, 0.5% NaOCl, Ozonated water, and 2 control groups. The root canals were irrigated with 20 mL of the selected solution for an uninterrupted period of 20 minutes, the irrigant solution was maintained resting for 5 minutes and then, samples were collected from root canals (S2). The data was evaluated using Kruskal Wallis and Wilcoxon tests (P<0.05). Results: The NaOCl group had lower microbial counts (P<0.05) and a higher percentage of microbiological reduction than the other groups (P<0.05). Conclusion: It was determined that ozonated water was not a suitable substitute for NaOCl.