This study was done to evaluate the antibacterial effect of chitosan nano-particles (CNPs) root canal irrigant as a new alternative to Sodium hypochlorite (NaOCl) for disinfection of root canals inoculated with , with and without laser activation. Sixty single rooted human premolars were decoronated, prepared and had their apical foramina sealed. were incubated in the root canals for 15 days. The teeth were then randomly divided into two experimental groups ( = 30) according to the disinfection protocol used. In Group I: disinfection was performed using the irrigant solutions only (Saline, 2.5% NaOCl, CNPs). Whereas in Group II, disinfection was done using the same irrigants followed by Diode laser at (980-nm) at 2 W output for 5 × 5 s. Intra-canal bacterial samples were taken before and after canal disinfection to determine the CFU count. In group I, 2.5% NaOCl was as effective as CNP in eradication and significantly more effective than Saline ( = 0.008) in eradication of . In Group II, either 2.5% NaOCl or CNP in combination with diode laser irradiation showed a similarly high effect in bacterial eradication. Within the parameters used in this study, a combination therapy consisting of irrigation followed by diode laser irradiation should be utilized as an effective treatment modality for eliminating E. faecalis from root canal systems.