Background: Bile typically remains sterile within individuals possessing a normal biliary tract, but bacterial colonization arises with biliary obstruction. Infections often stem from ascending duodenal infection or bacterial translocation from the portal vein. Elevated common bile duct pressure due to obstruction, combined with infected bile, can lead to bacterial backflow into lymphatics, fostering cholangitis. In this study, 30 sequential bile samples were examined, scrutinizing the occurrence of bacteriobilia and comparing its incidence between benign and malignant etiologies. Aims and Objectives: Bacteriobilia typically manifests without symptoms. This study aimed to assess the prevalence of bacteriobilia in patients with obstructive jaundice undergoing endoscopic retrograde cholangiopancreatography (ERCP) at our institution, investigating associated risk factors. Furthermore, the study aimed to elucidate the microbiological profile of bile in obstructed biliary systems, make a comparative analysis of bacteriobilia frequency between benign and malignant obstructions, and delve into the drug sensitivity patterns of bacteria cultured from bile at our center. Materials and Methods: In a prospective, observational, and diagnostic study conducted at the Department of Digestive Health and Diseases, Government Peripheral Hospital, Anna Nagar, Chennai, from September 2011 to February 2012, with ethical committee approval and informed consent from all participants, we investigated 30 consecutive patients who underwent ERCP for biliary obstruction. The study cohort comprised 13 males and 17 females. Following successful biliary cannulation, bile samples were collected and subjected to microbiological analysis. Results revealed positive bile cultures in 16 patients, with 10 exhibiting benign etiology and 6 malignant. Gram-negative organisms, predominantly Klebsiella, were identified, displaying favorable susceptibility to cephalosporins while demonstrating resistance to ciprofloxacin. Results: Bile cultures yielded positive results in 16 patients, with 10 cases attributed to benign etiology and 6 to malignant etiology. The cultured organisms were predominantly Gram-negative, with Klebsiella exhibiting significant growth. Notably, these organisms exhibited favorable susceptibility to cephalosporins while displaying resistance to ciprofloxacin. Conclusion: Bile culture serves as a valuable tool for prompt diagnosis and pre-emptive management of complications. Gram-negative bacilli infections prevail within obstructed biliary systems. This study underscores the pivotal role of routine bile sampling during ERCP in obstructed biliary systems, offering predictive insights to avert complications like cholangitis.