Objective To evaluate the antibacterial effects of the combination of calcium hydroxide and new triple antibiotics against Enterococcus faecalis (E. faecalis). Methods Eighty single rooted premolar teeth (intact and normal development) were included in this study. E. faecalis infected model was established in vitro. Model teeth were divided randomly into five groups with 16 teeth in each group, and the teeth in the five groups were treated using normal saline (NS group), calcium hydroxide (CH group), conventional triple antibiotics (mixed with metronidazole, minocycline and ciprofloxacin, TAP group), new triple antibiotics (mixed with ornidazole, minocycline and ciprofloxacin, NTAP group) or new triple antibiotics in combination with calcium hydroxide (NTAP+CH group), respectively, as intracanal medicament. The colony-forming units were counted at day 3 and day 7 after operation using colony-counting method. Scanning electron microscope was used to confirm the bacterial counting results. Results The colony-forming units on day 3 and day 7 of post-operation in the NTAP and NTAP+CH groups were both significantly lower than those in the TAP and CH groups (both P<0.05). Compared with the NTAP group, the NTAP+CH group showed a significant decrease in colony-forming units on day 3 of post-operation (P<0.05), but there was no significant difference on day 7 between the two groups (P>0.05). Scanning electron microscope results showed fewer bacteria on the root canal walls in the NTAP and NTAP+CH groups than the other three groups. Conclusion Antibacterial effect of new triple antibiotic used alone or in combination with calcium hydroxide for E. Faecalis is superior to conventional triple antibiotics and calcium hydroxide. So new triple antibiotic used alone or in combination with calcium hydroxide can be considered as an effective alternative intracanal medicament.