Objective: This study aimed to assess the frequency of positive bile cultures in patients undergoing cholecystectomy at three training hospitals in Tehran in 2009 and 2010. Methods and Materials: This was an observational, descriptive-analytical cross-sectional study. Patients undergoing cholecystectomy at the hospitals above during 2009 and 2010 were included in the study. Results: Among the patients included in the study, 29% had positive bile cultures. The most common germ found was E. coli (55.2%), followed by Enterobacter (13.8%), Klebsiella (10.3%), gram-positive bacteria (10.3%), proteus (6.9%), and pseudomonas (3.4%). Conclusions: Based on the results of this study and other similar studies, it can be concluded that positive bile culture is a common finding in patients undergoing cholecystectomy. Therefore, it is recommended to perform antibiotic prophylaxis in these surgeries.