Introduction: The aim of this study was to evaluate the antibacterial efficacy of Vitex negundo Linn. extract as root canal irrigant against Enterococcus faecalis and its penetration into root dentin. Methods and Materials: Forty single rooted premolars were randomly divided into 4 groups: 3% Sodium hypochlorite (NaOCl), 2% Chlorhexidine (CHX) , 100mg/ml Vitex negundo Linn. and saline as control all mixed with Rhodamine B dye. Test samples were analysed for bacterial count before and after irrigation using absorbent paper points and the colony forming units were recorded and measured. Sectioning of the samples was performed at three levels 3mm,6mm,9mm from apex and then these samples were analysed using confocal laser scanning microscopy for penetration depth of the irrigant within the dentinal tubules. Paired t-test and ANOVA test were used to perform statistical analysis with level of significance set at 0.05 Results: The mean CFU/ml count of Enterococcus facealis reduced significantly in all the groups post irrigation. All the irrigants showed maximum penetration depth at coronal third level compared to middle and apical third level respectively. The penetration depth of NaOCl group was better when compared to CHX group and Vitex negundo Linn. group but the difference was statistically not significant. Conclusion: Although 3% NaOCl was the most effective irrigant, all agents exerted acceptable antimicrobial activity against Enterococcus faecalis and penetration depth within tubules of dentin.