Background: Chronic calculous cholecystitis frequently involves bacterial colonization of bile, contributing to recurrent inflammation and postoperative complications; identifying pathogens and antibiotic sensitivity remains clinically important. Objective: To determine bacteriological profiles of gallbladder bile and analyze antimicrobial susceptibility patterns among patients undergoing cholecystectomy for chronic calculous cholecystitis. Methods: This cross-sectional descriptive study included 100 consecutive patients undergoing cholecystectomy at Rajshahi Medical College Hospital between January and December 2019. Demographic, clinical, and microbiological variables were recorded using a structured data sheet. Bile samples were cultured and tested for antibiotic sensitivity following CLSI guidelines. Data were analyzed using SPSS version 23, employing chi-square and Student’s t-tests with statistical significance set at p<0.05. Results: The mean age was 37.73 ± 5.19 years (range 28–69). Females predominated (62% vs 38%, ratio 1:1.63). Urban residence accounted for 58%, while 71% had normal nutritional status (p = 0.0001). Mixed gallstones were most common (62%), followed by cholesterol (30%) and pigment stones (8%). Bile culture was positive in 41% of patients. Among isolates, E. coli (39.02%) was predominant, followed by Pseudomonas aeruginosa (26.83%), Klebsiella (19.51%), and S. epidermidis (9.76%), while S. aureus and Salmonella typhi each accounted for 2.44%. Culture positivity was significantly associated with mixed stones (87.8%, p = 0.0001). Antibiotic sensitivity showed highest response to imipenem (100%), followed by cefuroxime (95.12%), gentamicin (90.24%), ciprofloxacin (85.37%), and ceftazidime (80.48%). Ceftriaxone resistance reached 17.08%. Conclusion: Most bile cultures remain sterile, yet significant bacterial colonization occurs in chronic calculous cholecystitis. E. coli predominates, and imipenem shows highest efficacy while ceftriaxone resistance raises concern.