Objective: To assess the antibacterial efficacy of chitosan nanoparticles (CSNPs), calcium hydroxide (Ca(OH)2) and double antibiotic paste (DAP) against 3-week-old Enterococcus faecalis biofilm. Materials and methods: Sixty extracted mandibular premolars were standardised to a specimen length (14 mm), instrumented and, after sterilisation, inoculated with E. faecalis and incubated for 3 weeks. Biofilm formation was confirmed by scanning electron microscopy in four randomly selected specimens. The remaining specimens (n = 56) were divided into four groups (n = 14): Group 1, 20 mg/ml CSNPs in methylcellulose; Group 2, 35% Ca(OH)2 in methylcellulose; Group 3, 1 mg/ml DAP in methylcellulose; and Group 4, methylcellulose gel (control group). Each group was further divided into two equal subgroups (n = 7) and incubated for 7 or 14 days. After each test period, a sample of the biofilm was collected from each specimen, plated, and the antimicrobial efficacy assessed via colony forming units count (CFU/ml). Results: All the antibacterial agents evaluated resulted in complete eradication of E. faecalis after 14 days. However, Ca(OH)2 was the only medicament that provided a significant bacterial count reduction after 7 days (P 0.05). No statistically significant difference was found between Ca(OH)2, CSNPs, and DAP (P ≥ 0.05). Conclusions: A 14-day period of dressing with CSNPs, Ca(OH)2, or DAP resulted in effective antibacterial action on the 3-week-old E. faecalis biofilm. Keywords: calcium hydroxide, chitosan nanoparticles, double antibiotic paste, endodontic regeneration, Enterococcus faecalis