Objective: The antimicrobial efficacy of three disinfection solutions: 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) and Listerine mouthwash were investigated as routine chair-side gutta-percha (GP) disinfection reagents. Design: four groups of gutta percha points were contaminated with E. faecalis bacteria then disinfected by immersion in different solutions (5.25% sodium hypochlorite, 2% chlorhexidine gluconate, Listerine mouth wash and distilled water as control) after 1 and 7 days culturing periods. The antibacterial efficacy of these disinfection solutions was evaluated by using colonies per units (CPU) Methods: Forty GP cones (F3 Dentsply) were sterilized with ethylene oxide gas before immersed contamination within broth media containing E. faecalis for 20 min. Cones then divided them equally into four groups (n = 10) based on the treated disinfection solution, whilst one group received no disinfection treatment (control group). Each cone was then immersed into broth media within separated Eppendorf tubes for 3 min before 100 μl was taken to be cultured in an agar plate for 24h. The remaining broth media tubes were incubated for 7 days at 37℃ before taking another 100 μl to be cultured in new agar plates. CPU for E. faecalis growth were calculated for the two period cultured plates: immediately after GP disinfection (day 0) and after 7 days incubation. Results: All groups except control showed low E. faecalis colonies growth. The colony-forming units (CFU) of E. faecalis on day 0 was zero growth in NaOCl and CHX disinfected groups and much higher (14) within Listerine group. On day 7, only NaOCl group still have complete inhibition for E. faecalis colonies growth. CHX antimicrobial potency was decreased with time, while CFU for Listerine group decreased after 7 days incubation. Conclusion: The NaOCl was the best disinfection for gutta-percha cones, followed by CHX and Listerine mouthwash