Background and Objectives: This ex vivo study aimed to assess the antibacterial efficacy of diode laser, non-activated sodium hypochlorite (NaOCl), NaOCL activated by 980 nm diode laser or Passive Ultrasonic Irrigation (PUI) technique for elimination of Enterococcus faecalis (E. faecalis) from the infected root canals. Materials and Methods: Root canals of 90 single-rooted human premolars were instrumented with ProTaper rotary system. The teeth were sterilized and their root canals were inoculated with E. faecalis except for five samples, which served as negative controls. The teeth were incubated at 37°C for three weeks and were then randomly divided into four groups (n=20). Group A root canals were rinsed with 5.25% NaOCl for 60 seconds; group B root canals underwent diode laser (980 nm) irradiation (1.5 W, CW, 4*10 s) while filled with 5.25% NaOCl; group C root canals were subjected to passive ultrasonic irrigation (PUI) with 5.25% NaOCl for 60 seconds; group D root canals underwent diode laser irradiation (1.5 W, CW, 4*10 s). Infected root canals in the positive control group (n=5) did not undergo any intervention. Samples were taken from the intracanal bacteria and cultured to determine the number of colony forming units (CFUs). Data were analyzed using the Kruskal-Wallis test. Results: among the treatment groups, the lowest bacterial count was noted in sodium hypochlorite group agitated with ultrasonic while the highest bacterial count was noted in diode laser group and this difference was statistically significant (P=0.004). No other significant differences were noted (P>0.05). Conclusion: All the tested methods are suitable for elimination of E. faecalis from the root canal system. However, it appears that NaOCl, with/without agitation, is more effective than diode laser for decreasing intracanal infection