Higher morbidity and mortality is seen with bacteraemia caused by Biliary tract infection. Due to the presence of biliary sphincter, bile remains sterile despite presence of high concentration of bacteria in duodenum. Increased ductal pressure due to obstruction in biliary tree, can result in biliary stasis and bacterial multiplication resulting in cholangitis. 16% to 85% of bacteriobilia have been reported in various studies worldwide in patients of biliary tract infection. Govind Ballabh Pant institute of Postgraduate Medical Education & Research (GIPMER) is a super speciality hospital providing care to patients from all over India. This study was performed to identify the bacteria causing Biliary tract infection and study their resistant pattern. A total of 229 intraoperative bile aspirates from gastrointestinal surgery department were received in microbiology department of GIPMER and analysed. Identification and antimicrobial susceptibility testing was performed as per standard techniques. Bacterial growth was seen in 166(72.48%) out of 229 patients. Polymicrobial cultures 27(16.27%) were less frequent in comparison with Monomicrobial growth 139(83.73%). Commonest isolate was Escherichia coli (47.94%) followed by Klebsiella pneumonia (17.53%), Pseudomonas aeruginosa (16.49%), and Enterococcus spp. (11.34%). High resistance was observed against routinely used antibiotics by Gram negative bacteria, such as ciprofloxacin, ceftriaxone, piperacillin/tazobactam, and low resistance was observed against carbapenems and colistin. The study shows that analysis of bile can be valuable in preparing a better treatment strategy in biliary tract infection. Rapid development of multidrug resistance in gram-negative bacteria causing biliary tract infections remains the cause of concern.