Using the modern anaerobic transport media and meticulous culture techniques, 74 patients undergoing biliary tract surgery were studied. The biliary system was found to be sterile in 58 patients (78%). Fifteen patients had 35 isolates of aerobic and facultative bacteria. The most common ones were Klebsiella, Enterococcus, and Escherichia coli. The only anaerobe isolated was Clostridium perfringens. Eight of 17 patients (47%) with acute cholecystitis and five of 49 patients (10%) with chronic cholecystitis, harbored bacteria in the biliary system. This study suggests that anaerobes are rare in the human biliary system; therefore, if antibiotic therapy is considered, aerobic coverage should suffice.