Background: Cholecystectomy is currently a frequently performed operation. The most common reason for a Cholecystectomy is gallbladder stones. Usually the bile ducts are sterile. However, the presence of gallstones within either the gallbladder or biliary tree is associated with the bacterial colonization of the bile. Therefore, it is important to know the microbiological flora of the gallbladder before prophylactic antibiotics are given. Aim and Objectives: To evaluate the microbiological profile of bile from gall bladder in patients undergoing Cholecystectomy. Methods: Patients undergoing laparoscopic or open Cholecystectomy in SSG Hospital, Baroda between September 2018 and December 2019 were included in this study and bacteriological examination of bile were done of this patients bile. Results: In this study a total of 78 patients(54 female and 24 male) who met the inclusion crieteria were included both open as well as laparoscopic Cholecystectomy . Number of Patients with positive bile cultures were 19(24.36%) and those with negative culture were 59(75.64%). Escherichia coli was the most common isolated bacteria Among positive bile culture group (63.16%) and 15.38% Among all patients. Other organisms isolated were pseudomonas (3.85% Among all patients), Klebsiella (2.56% Among all patients), Coagulase Negative Staphylococcus and Staphylococcus Viridans (1.28%). Conclusion: In vast majority of patients the bile was sterile, and Escherichia coli was the most common isolated bacteria Among positive bile culture group.