Objective To clarify the bacterial spectrum and drug resistance of different biliary diseases through bile culture results. Methods Patients who underwent surgical treatment and retained bile for cultivation at the Chinese PLA General Hospital between January 2015 and December 2016 were retrospectively collected. Clinical data such as bile bacterial culture and antibiotic sensitivity results, surgical reasons, and perioperative complications were recorded. Results A total of 272 patients were included, including 142 males and 130 females, aged (53.4 ± 14.1) years old. Intrahepatic and extrahepatic bile duct stones were the most common surgical cause, accounting for 32.4%. The positive rate of bile culture in benign diseases was 78.7%, which was higher than that in malignant diseases (48.5%). The infection related complications (30.0% vs. 6.7%), bile leakage rate (20.8% vs. 6.7%), and poor wound healing rate (24.0% vs. 0.0%) in the bile culture positive group were higher than those in the bile bacteria culture negative group (P<0.05). Among 183 patients with positive bile bacterial culture, a total of 294 strains of pathogenic bacteria were detected. There were 96 patients with single bacterial infection, 66 patients with two bacterial infections simultaneously, 18 patients with three bacterial infections, and 3 patients with four or more bacterial infections. Escherichia coli was the most common bacterium, accounting for 17.0%. There were differences in the positive rate of bile culture among patients with different etiologies (P<0.05). There were significant differences in the sensitivity rate of Enterococcus faecalis and Enterococcus faecalis for many antibacterial drugs. Conclusions There are differences in the positive rate of bacterial culture in the biliary tract of patients with different etiologies, and there is a possibility of mixed infection. It is necessary to select appropriate antibiotics for empirical treatment based on different etiologies. The use of antibiotics should be changed in a timely manner based on the results of bile culture.