Background To determine the antibacterial effect of chitosan-propolis nanoparticle (CPN) as an intracanal medicament against Enterococcus faecalis biofilm in root canal. Methods 240 extracted human teeth were sectioned to obtain 6mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into eight groups ( n=30 ) according to the intracanal medicament placed: group I: saline, groupII: chitosan, group III: propolis100 µg/ml (P100), group IV: propolis 250 µg/ml (P250), group V: chitosan-propolis nanoparticle 100µg/ml (CPN100), group VI: chitosan-propolis nanoparticle 250 µg/ml (CPN250), group VII: calcium hydroxide(CH) and group VIII: 2% chlorhexidine (CHX) gel. Dentine shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of day one, three and seven. The non-parametric Kruskal Wallis and Mann-Whitney tests were used to compare the differences in reduction of CFUs between all groups and probability values of P < 0.05 were set as the reference for statistically significant results. The scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to CPNs. The effectiveness of CPNs were also evaluated against E. faecalis isolated obtained from patients having failed root canal treatment. Results Reduction in the number of colony‐forming units was statistically significant in all groups compared to saline (p <.05). On day one and three, at 200 and 400-μm, CPN250 showed significant reduction of CFUs compared to all other groups (p <.05), while CPN100 was significantly better than other groups (p <.05) except CPN250 and CHX. On day seven, at 200-μm CPN250 showed significant reduction of CFUs compared to all other groups (p <.05) except CPN100 and CHX, while at 400 μm CPN250 showed similar effectiveness as CPN100, CH and CHX. SEM and CLSM images also showed the maximum reduction of E. faecalis with CPN250. Conclusion CPN250 was the most effective in reducing E. faecalis colonies on day one, three at both depths and at day seven CPN250 was equally effective as CPN100 and CHX.