Statement of Problem: The main goal of root canal therapy is total elimination of bacteria from root canals. Studies have shown that the cleaning and shaping of root canals can not complexly eliminate these bacteria from the root canals. Obturation may kill the bacteria due to entrapment of them into the canals and prevent their access to nutrition. On the other hand, using of Ca (OH)2 before obturation may help to promise a better reduction of bacteria from root canals.Purpose: The purpose of this study is an in vitro evaluation of antibacterial effect of obturaiton solely, or with using Ca (OH)2 as an intracanal dressing on E.F. in infected human root canals.Materials and Methods: For this experimental study, 50 single rooted human teeth without significant curvature were chosen. After cutting the crowns and preparing root canals, the samples were sterilized and then contaminated with Entrococcus faecalis bacteria for 5 days. The samples were divided randomly into 4 groups. The first experimental group which included 20 teeth, were filled by Ca (oH)2 for one week before obturation by lateral condensation technique. The second experimental group (20 samples) were obturated by ZOE sealer and gutta percha with lateral condensation for 2 weeks. Five roots considered as positive control were contaminated with bacteria but were not obturated, and 5 other roots were obturated after sterilization without contamination as negative control group. All samples were incubated in 35oC and 10% moisture for 2 weeks. Then the coronal and apical part of roots were removed and the dentin powder were collected from the middle parts by drilling the canals in sterile condition. Two mg of dentin powder was weighed from each root and cultured in blood agar for 48 hours. The results were recorded and compared with each other by Chi-Square test.Results: All samples were positive in culture of positive control group, and negative in culture of negative control group. First experimental group did not show any positive growth after the incubation period and there was only one positive culture in the second experimental group.Conclusion: The findings of this study illustrated no significant difference between the two experimental groups. In other words, the obturation of canals with gutta percha and sealer merely could kill the E.F. in the infected root canals and application of Ca (OH)2 before obturation was not necessary for disinfection of canals.Key words: Bacterial Growth, Calcium Hydroxide, Entrococcus faecalis