Background: Oral microorganisms, which are usually opportunistic pathogens invade a root canal containing necrotic tissue and establish an infectious process causing primary endodontic infections. When root canal remains infected for long periods, the number of facultative anaerobic bacteria increases. The most common facultative anaerobic Gram-positive coccus cultured from non-healing endodontic cases is Enterococcus species. Aims and Objectives: The present study was done to evaluate the antimicrobial efficacy of Tridax procumbens, Aegle marmelos, and 5% sodium hypochlorite against Enterococcus faecalis biofilm formed on the tooth substrate. Materials and Methods: Extracted human teeth were biomechanically prepared, vertically sectioned, placed in the tissue culture wells exposing the root canal surface to E. faecalis to form a biofilm. At the end of the 3 rd and 6 th weeks, all groups were treated for 10 min with the test solutions and control and were analyzed qualitatively and quantitatively. Results: Qualitative assay with 3-week biofilm showed complete inhibition of bacterial growth with T. procumbens, A. marmelos and NaOCl, except saline, which showed the presence of bacterial growth. In quantitative analysis, Aegle and saline-treated tooth samples showed 1.344 ±123 CFU/mL and 186.4 × 10 9 ± 4.1 × 10 9 CFU/mL (mean and standard deviation), respectively. Qualitative assay with 6-week biofilm showed growth when treated with Tridax and marmelos whereas NaOCl has shown complete inhibition. All treated groups have shown a significant reduction of the bacterial population compared with the control group, and NaOCl showed 100% reduction. Conclusion: Nearly 5% sodium hypochlorite showed maximum antibacterial activity against E. faecalis biofilm formed on tooth substrate. T. procumbens, A. marmelos also showed statistically significant antibacterial activity.