Background: the present study was undertaken for evaluating Anti–Microbial Efficacy of Ozonated Water on Microorganisms Present in Root Canal System. Materials & methods: 45 patients with primary endodontic infection were treated with ozonated water ¬and samples were analysed for microorganisms. All the results were recorded in Microsoft excel sheet and were analysed by SPSS software. Results: patients with microbes after treatment with ozonated water reduced from 97.78% to 62.22%. Gram Negative bacteria reduced from 82.22% and 51.11%. Gram positive bacteria reduced from 15.56% to 11.11%. aerobic bacteria reduced from 66.67% to 42.22%. anaerobic bacteria reduced from 31.11% to 20%. Conclusion: Ozonated water is an effective but not potent antimicrobial irrigating solution and is more effective against anaerobic than aerobic bacteria.