El-Mosalamy., et al.Objective: The purpose of this In-vitro study was to evaluate the antibacterial efficacy of sodium hypochlorite and nano-chitosan irrigations (CNPs) with two different irrigation techniques, conventional syringe irrigation (CSI) and Passive ultrasonic irrigation (PUI) on Enterococcus Faecalis biofilm reduction inside the infected dentinal tubules using confocal laser scanning microscope (CLSM).Methods: Twenty eight teeth samples were bio-mechanically prepared, sterilized, and inoculated with E. faecalis (ATCC 29212) for 3 weeks. Specimens were then randomly divided into four groups (n = 7) according to the final irrigation protocol as follows: Group I, CSI with 2.6% NaOCl; Group II, irrigation with 2.6% NaOCl + PUI; Group III, CSI with 0.5% CNPs; while in Group IV, irrigation with 0.5% CNPs + PUI. Samples were then longitudinally sectioned to obtain a 4x2 mm sized sections from the apical 1/3 of the roots. E. faecalis biofilm reduction was finally evaluated using CLSM and at three different 100 µm areas representing 3 depths inside the dentinal tubules. Results: None of the four experimental groups could result in complete elimination of E. faecalis from the dentinal tubules. Group II (NaOCl+PUI), III (CNPs), and IV (CNPs + PUI) all showed significantly higher biofilm reduction when compared to Group I (NaOCl) (P-value < 0.001). However there was no statistically significant difference between the three groups (P-value > 0.05). Conclusion:Active and passive CNPs irrigations showed superior antibacterial efficacy in comparison to NaOCl, and were on bar with PUI activated NaOCl, no improvement in the antibacterial efficacy could be obtained from using PUI to activate CNPs irrigation.