The aim and objective of this study are to evaluate and compare the antimicrobial efficacy of ozonized calcium hydroxide, calcium hydroxide with chlorhexidine and double antibiotic paste when used as intracanal medicament. Materials and methods: 30 extracted single-rooted, single canal human teeth were selected for the study. Teeth were decoronated and the length was standardized to 13mm. After cleaning and shaping of the canals, teeth were inoculated with E. fecalis and samples were collected before placement of medicament. Teeth were then divided into 3 groups. Group A: Calcium Hydroxide + Chlorhexidine; Group B: Calcium Hydroxide + Ozonized water & Group C: Double antibiotic paste. Microbial samples were collected after 48 hours and were evaluated. Results: Kruskal Wallis test followed by Dunns Post hoc test were used for analysis, with level of significance P﹤0.5. Group A demonstrated significantly lesser CFUs/ml values as compared to Group B & Group C & the difference was statistically significant at p=0.04 & p=0.001 respectively. This was followed by Group B showing relatively lesser mean CFUs/ml as compared to Group C and however the mean difference was not statistically significant [p=0.17]. This infers that the mean CFUs/ml value was significantly lesser in Group A, followed by Group B and higher in Group C Conclusion: Calcium hydroxide mixed with chlorhexidine followed by calcium hydroxide mixed with ozonised water is an effective intracanal medicament against E.fecalis when compared to double antibiotic paste. Clinical Significance: ozonated water can be considered a vehicle to carry calcium hydroxide as an intracanal medicament