Background Gallbladder and biliary tract infections are diseases with high mortality rates if they are not treated properly. Microbiological evaluation of perioperatively collected samples both ensures proper treatment of patients and guides empirical treatment due to the determination of microorganism susceptibility. Aims This study aimed to isolate the microorganisms in bile cultures from patients who underwent cholecystectomy and to determine sensitivity results of these microorganisms. Methods This study was a multi-center and prospective design, included 360 patients, and was performed between 2019 and 2020. Culture results of bile taken during cholecystectomy were evaluated. Results Bacterial growth was found in the bile cultures of 84 out of 360 (23.3%) patients. Patients were divided into two groups according to whether they had risk factors for resistant microorganisms or not. While Escherichia coli (n = 11, 13%), Enterococcus spp. (n = 8, 9.5%), and Enterobacter spp. (n = 4, 4.7%) were detected most frequently in patients without risk. Staphylococcus spp. (n = 17, 20.2%), Enterococcus spp. (n = 16, 19%), and E. coli (n = 8, 9.5%) were the most frequently found microorganism at-risk patients. In multivariate analysis, bile culture positivity was found higher in patients who had history of biliary disease (p = 0.004), operation performed concurrently with a cholecystectomy (p = 0.035), and high rate of polymorphonuclear leukocytes (PNL) in total leukocyte count (p = 0.001). Conclusions Our study shows that when starting empirical antibiotic treatment for bile ducts, whether patients are at risk for the development of resistant bacterial infection should be evaluated after which antibiotic selection should be made accordingly.