Aim: To investigate the antimicrobial activity 2% Chlorhexidine gel (CHX), Aloe vera, Propolis, Septilin and Calcium hydroxide [Ca (OH) 2] on Enterococcus faecalis infected root canal dentine at two different depths (200 µm and 400 µm) and three time intervals (day 1, 3 & 5). Methodology: One hundred and eighty extracted human teeth were infected for 21 days with E.faecalis. Samples were divided into six groups. Group I (Saline) (Negative control), Group II (Propolis), Group III (2% CHX), Group IV (Aloe vera), Group V (Calcium hydroxide), Group VI (Septilin). At the end of 1, 3, and 5 days, the remaining vital bacterial population was assessed. Dentine shavings were collected at two depths (200 µm and 400 µm), and total numbers of colony forming units were determined. The values were analysed statistically with oneway analysis of variance followed by Tukey multiple comparison test. The paired t-test was used to check for differences in growth at different time intervals within groups and for differences at the two depths (P < 0.01). Results: The number of colony-forming units was statistically significant in all groups compared to the control group (Saline). Group III (CHX) and Group VI (septilin) (100%) produced better antimicrobial efficacy followed by aloe vera (78.94%), propolis (66.7%), calcium hydroxide (58.5%). There was significant difference between aloe vera and propolis and no significant difference between data at 200 µm and 400 µm. Conclusion: Septilin and Aloe vera were effective against E.faecalis in dentine of extracted teeth.