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Chitosan and its derivatives have been increasingly used for bacteriostasis. To date, the effect of chitosan and N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) on Enterococcus faecalis (E. faecalis) associated with endodontic infection has remained to be determined. Chitosan and HTCC were serially diluted with double-distilled water (DDW) or PBS at concentrations of 20-2,500 µg/ml. Various strains of E. faecalis (American Type Tissue Collection no. 29212, as well as isolated strains P25RC and P52Sa) in plankton were adjusted to an optical density at 600 nm of 0.10 and treated with chitosan or HTCC. A colony-forming unit assay was used to determine the concentration of residual bacteria after treatment. Furthermore, E. faecalis biofilms were cultured on coverslips and treated with chitosan or HTCC. The coverslips were rinsed, stained using Live/dead® BacLight™ bacterial viability kit and observed under an inverted fluorescence microscope. In addition, biofilms on dentine blocks were prepared and observed under a scanning electron microscope. MC3T3-E1 pre-osteoblasts were seeded on 96-well plates and treated with chitosan or HTCC at various concentrations. The cytotoxicity of chitosan and HTCC on MC3T3-E1 pre-osteoblasts was detected using a Cell Counting Kit-8 assay after 24, 48 and 72 h of treatment. The results revealed that the final minimum bactericidal concentrations (MBC) of chitosan and HTCC dissolved in DDW were 70 and 140 µg/ml, respectively. Chitosan and HTCC in DDW exerted a significantly greater antibacterial effect as compared with that in PBS (P<0.05). At the MBC, chitosan and HTCC in DDW, but particularly chitosan, had a significant antibacterial effect on E. faecalis biofilm. Chitosan exhibited no cytotoxicity to MC3T3-E1 pre-osteoblasts at a concentration of <625 µg/ml, while HTCC inhibited the proliferation of the cells in the concentration range of 39-10,000 µg/ml. In conclusion, chitosan and HTCC exhibited prominent antibacterial properties on E. faecalis in the planktonic state and as a biofilm via charge interaction, indicating their potential for application in root canal disinfection and fillings. [ABSTRACT FROM AUTHOR]