Statement of Problem: The main goal of endodontic treatment is eradication of microorganisms and their byproducts from the root canal system. The use of chemical irrigants during chemo-mechanical canal preparation is important for disinfection and cleaning of the canal system.Purpose: The aim of this in vitro study was the assessment of bacterial growth after contact with several concentrations of essential oil of Zataria multiflora in varying time intervals.Materials and method: The essential oil of Zataria multiflora were serially diluted 8 folds and a fixed volume of the stationary phase  culture of E.faecalis was added to each diluted solution, for varying intervals of 1, 5 and 15 minutes. 100µl of the contents of each tube were transferred to SF broth and incubated for 48h at 370c. Results of bacterial growth were recorded at the end of the incubation period.Results: 2% solution of essential oil of Zataria multiflora at all intervals inhibits growth of E.feacalis. 1% solution of it showed antibacterial effect in 5, and 15 minutes. The concentrations under 0.5% dilution of Zataria multiflora did not eliminate E.faecalis at any time intervals.Conclusion: It seems that 1% and 2% solutions of essential oil of Zataria multiflora were effective at destroying E.faecalis. Application of this plant essential oil may be recommended for root canal irrigant following extensive ex-vivo and in-vivo experiments.Key words: Essential oil of Zataria multiflora, Enterococcus faecalis, antimicrobial activity