Background: Gallstone disease remains one of the most common medical problems leading to surgical intervention. The presence of gallstones within either the gallbladder or biliary tree is associated with the bacterial colonization of the bile. In the vast majority of cases, infection is due to bacteria originating from the biliary tract. The incidence and type of bacteria involved and their susceptibility to antibiotics can be accurately predicted by bacteriological examination of the bile and a suitable antibiotic regimen can then be selected to minimize post-surgical infections. Therefore, it is important to know the microbiological ora of the gallbladder before prophylactic antibiotics are given. Materials and Methods: Study design: Study area: A descriptive study. Ramakrishna Mission Seva Pratishthan Hospital, Kolkata. from October 2017 to April 2019 (18 months). Sample size: 300 patients admitted in the Department of General Surgery undergoing Laparoscopic/Open Cholecystectomy. Inclusion criteria: Patients diagnosed with Chronic Calculous Cholecystitis belonging to any age or sex. Exclusion criteria: Patients with acute cholecystitis, empyema gallbladder, mucocele of gallbladder, gallstones along with bile duct stones. Methodology: Intraoperatively, 2ml bile from gallbladder was aspirated and sent for microbiological analysis. Each specimen was inoculated on two blood agar plates and one MacConkey agar plate. Growth was conrmed from colony characteristics, by Gram's staining, biochemical reactions and any special tests required conrming the particular bacterium. Antibiotic sensitivity was done by Kirby-Bauer method. Results: Bile Culture was positive in 39(13%) patients, of which majority were Females(61.5%) and majority were in the 61-80 age group for both. The most common organism cultured in Bile was E.coli(59%), followed by Klebsiella (25.6%). Majority were sensitive to Cefuroxime (28.2%), followed by Ciprooxacin (20.5%). Metronidazole was found sensitive in 12.8% cases whereas, Levooxacin, Meropenem and Piperacillin-Tazobactam were sensitive in 10.3%, 12.8 and 5.1% cases. 3 cases (7.7%) were found to contain microorganisms resistant to commonly used antibiotics. Conclusion: Biliary cultures were positive in 13% of cases with enterobacteria being the most frequent isolate.