Statement of Problem: Given the importance of bacteria in the development of periradicular lesions, the eradication of the root canal infection is paramount in endodontic treatment. The use of chemical irrigants during chemomechanical canal preparation is important for disinfection and cleaning of the canal system. Purpose: The aim of this in vitro study was the evaluation of effectiveness of 1% and 2% essential oil of Zataria multiflora as an irrigant in the elimination of Enterococcus faecalis from the infected root canals.Materials and Methods: The antibacterial effects of the irrigants in vitro were examined after 15 min, in freshly extracted human teeth with single roots, the canals of which were infected by E.faecalis.2ml of 2.5% NaOCl2ml of 1%   Zataria multiflora2ml of 2%   Zataria multiflora2ml of sterile normal saline( as control) Paper points used to sample bacteria from the root canals were transferred to tubes containing 5ml of brain heart infusion (BHI) broth. The tubes were incubated and the appearance of broth turbidity was indicative of bacteria remaining in the root canal. The data obtained were analyzed statistically for differences using the chi-squared test, comparing pairs of groups ) p < 0.05(.Results: There was no statistically significant differences between the experimental groups ( p >0.05). However, the experimental irrigants were significantly more effective than the saline solution (control group) in disinfesting the root canal.Conclusion: It seems that essential oil of Zataria multiflora was effective at killing E.faecalis. Application of this plant essential oil as root canal irrigants may be recommended following extensive ex vivo and in vivo experiments. The findings recommend further studies of the properties of Zataria multiflora such as toxicity, tissue solving ability, and biocompatibility to be applied for clinical use.Key word: Zataria multiflora, root canal irrigant, Enterococcus faecalis