Background and objectives: Bacterial infection of biliary tract may cause severe inflammatory response or sepsis. An immediate bile culture and appropriate antibiotic administration are important to control the biliary tract infection. The objective of the study was to study the microbial profile and antibiotic sensitivity pattern in patients with biliary tract infection. Materials and methods: Fifty suspected cases of biliary tract infection admitted to the Department of Gastroenterology were enrolled. Bile samples from these patients were aseptically collected and sent to the Department of Microbiology. Samples were processed in automated BACTEC or BacT/ALERT system. Further identification and antimicrobial susceptibility testing was done by VITEK-2 system. Results: Of the 50 suspected cases of biliary tract infection, the majority were male patients and in the age group of 51-60 years. Growth was obtained in 22 (44%) bile samples. The organisms obtained were Escherichia coli (40%), Klebsiella spp. (20%), Pseudomonas spp. (16%), and coagulase-negative Staphylococci (8%). All Gram negative isolates were susceptible to tigecycline and colistin. A high susceptibility was seen to amikacin and carbapenems while low susceptibility was seen to others. All the Gram positive organisms were sensitive to vancomycin, teicoplanin and linezolid with low sensitivity to penicillin. Interpretation and conclusion: E. coli is the most common organism isolated from bile. Antimicrobial sensitivity patterns require a revision of empiric antibiotic therapy policy in cholangitis. Early detection and determination of antimicrobial susceptibility pattern is important to reduce the mortality and morbidity associated with bile fluid infections.