Aim: To Compare the antibacterial efficacy of Chlorhexidine, nano-chitosan and their combination against enterococcus faecalis (E. faecalis) with and without ultrasonic activation.Patients and Methods: 110 extracted teeth were divided into 6 groups according to the antibacterial agent used; 1: Control group, 2: chitosan, 3: chitosan+2% chlorohexidine, 4: 2% chlorohexidine, 5: chitosan extra-strength, 6: chitosan extra-strength+ 2% chlorohexidine. Each group was subdivided into two subgroups, with and without ultrasonic activation (n=10). Microbial samples were collected from all the root specimens and colony forming units were counted and transformed into log CFU. The collected data were statistically analyzed using Kruskal Wallis test and pairwise Mann -Whitney U test with Bonferroni correction. Results:The control group showed the highest bacterial count while CHX with ultrasonic activation group showed the lowest bacterial count. There was a significant difference in bacterial count between the 6 groups (p< 0.001). Pairwise comparison revealed that CHX, chitosan+ CHX and chitosan extra-strength +CHX groups with ultrasonic activation showed significantly lower bacterial count than chitosan with ultrasonic activation group, chitosan extra-strength without ultrasonic activation group and the control group. Chitosan extra-strength with ultrasonic activation group, chitosan extra-strength + CHX, chitosan, chitosan + CHX and CHX groups without ultrasonic activation showed no significant difference in bacterial count from all other groups.Conclusions: Ultrasonic activation improves bacterial elimination, CHX with ultrasonic activation showed higher antimicrobial effects against E. faecalis among all tested groups.