Abstract : The main goal of root canal treatment is to clean and disinfect the root canal system through elimination of bacteria and removal of necrotic tissue with mechanical instrumentation and irrigation. The purpose of this study was to determine the antibacterial efficacy of sodium hypochlorite and chitosan oligosaccharide against Enterococcus faecalis (E. faecalis) in endodontic treatment with different instrumentation. Twenty-four single-root mandibular premolars were divided into four groups. Teeth were divided into four groups, namely A (sample irrigated with 2.5% NaOCl and 17% EDTA and prepared with F-One Blue), B (sample irrigated with 2.5% NaOCl and 2% Chitosan Oligosaccharide and prepared with F-One Blue), C (sample irrigated with 2.5% NaOCl and 17% EDTA and prepared with One-Curve), D (sample irrigated with 2.5% NaOCl and 2% Chitosan Oligosaccharide and prepared with One-Curve). Then, an examination of E. faecalis biofilm and residual dentin was carried out. The data obtained was subjected to a One-way ANOVA statistical test and continued with the LSD Post Hoc test. The results of the One-way ANOVA test showed that there was a significant difference in the growth of E. faecalis biofilm between the treatment groups. From the research results it was found that Groups B and D had a very good ability to reduce the mass of E. faecalis biofilm. Based on the scatter analysis, the group with root canal irrigation using 2.5% NaOCl and 2% COS and instrumentation with F-One Blue showed the best ability to eliminate E. faecalis and inhibit the formation of residual dentin.