The aim and objective of this study was to compare the antimicrobial efficacy of 0.2% chitosan, 3% sodium hypochlorite, 2% chlorhexidine against Enterococcus faecalis, alone and in conjunction with diode laser. Materials and methods: The root canals of 72 extracted intact human single-rooted teeth with single canals were prepared, and E. faecalis was incubated in the root canals for 7 days. The teeth were then randomly divided into the following four experimental groups: group I: Saline, group II: 0.2% Chitosan, group III: 3% Sodium hypochlorite, and group IV: 2% Chlorhexidine. These groups were further subdivided into three groups: (1) 10 mL irrigant only, (2) 10 mL irrigant, dried and irradiation with diode laser, (3) Diode laser was used for activation of irrigant solution. Samples were obtained from subgroups in each group and checked for turbidity. The effect of each irrigant was evaluated by counting the number of colony-forming units observed on inoculation with samples taken from the irrigated canal on bile esculin azide agar. The data thus obtained was recorded and put to statistical analysis. Results: Significant reductions were noted in E. faecalis colony counts in all groups (p < 0.05). The greatest reduction in colony count (0%) was noted in group IV followed by group II. Also, samples disinfected with diode laser after root canal irrigation showed less number of colony-forming units per mL as compared to the samples irrigated with root canal solutions alone or diode laser alone. Conclusion: Chitosan has the capability for use as an accessory for disinfection of the root canal system. The application of an 810-nm diode laser by itself did not have the adequate antimicrobial activity to be used as an adjunct in root canal therapy. Irradiation with diode laser ought to be used in conjunction with the irrigant to gain maximum antibacterial effect against E. faecalis.