Aim: To determine the antimicrobial efficacy of ozonated water and compare the antimicrobial activity of different irrigant solutions against E.faecalis and E.coli. Material and Method: After incubation, 21 contaminated roots were divided into 3 groups according to the irrigation regimen used i.e. Group A: Ozonated Water (7 teeth), Group B: 3% NaOCl (7 teeth) and Group C: Saline (7 teeth). All the teeth were handled with sterile gloves and sterile tweezers to prevent contamination. A sterile 5 mL syringe with 26 gauge needle was used to deliver irrigant into the canal. All experimental teeth were then flushed with distilled water to prevent potential carry–over of irrigants. Results: Results indicated that Group C showed highly significant difference from group A and Group B indicating that Group A and Group B were effective against both E.faecalis and E.coli. Results also show that there was no significant difference in efficacy of Group A and Group B. Conclusion: The results found that ozonated water has proved to be an alternative antimicrobial agent.