Cholelithiasis, choledocholithiasis and hepatolithiasis are common biliary tract diseases. These diseases may cause severe infection and/or sepsis. In addition to surgical treatments, prompt administration of appropriate antibiotic is important to control the biliary tract infection. The purpose of this study is to illustrate the bacteriology in biliary tract disease and provide information for antibiotic choices. From Jan 1991 to Aug 2000, 1394 patients including gallbladder (GB) stones, common bile duct (CBD) stones, intrahepatic duct (IHD) stones, GB polyps and biliary malignancy were subjects for this retrospective study. The overall positive rate of bile culture is 36% in this study while it was 25%, 66%, 67% and 9% for GB stones, CBD stones, IHD stones and biliary malignancy, respectively. A significantly higher (p = 0.001) positive culture rate was found for GB stones with acute cholecystits (47%) compared with that without inflammation (17%). Similarly, the culture rate for hepatolithiasis with acute cholangitis was higher than that without cholangitis (75% vs 51%, p = 0.011). Long-term external biliary drainage in biliary malignancy increased the risk of bacterial culture rate. For gallstone diseases, the most common organisms cultured were Gram negative bacteria (74%), in which Escherichia coli (36%) and Klebsiella (15%) were most commonly found, followed by Gram positive (15%) bacteria such as Enterococcus (6%), Staphylcoccus (3%), Streptococcus (2%). Bacteroides (5%) and Clostridium (3%) were occasionally found anaerobes (9%). Polymicrobial infection was encountered in 19%, 31% and 29% for patients with GB stones, CBD stones and IHD stones, respectively; frequency of mixed aerobic and anaerobic infection was 7%, 12% and 9%. In the current study, ampicillin in combination with sulbactam and aminoglycoside is still a suggestive empirical therapy. Antibiotic treatment should be adjusted based on later bacteriological cultures and clinical condition.