Background: Analyze the distribution and drug resistance of pathogenic bacteria that cause biliary tract infections in Nanjing, and provide evidence for the rational use of antibacterial drugs in clinical practice. Methods: Clinical strains isolated from bile specimens of patients suspected of biliary infection in the First Affiliated Hospital of Nanjing Medical University in 2019 were collected, The drug susceptibility criteria are based on the standards published by the National Standardization Committee of the US Clinical Laboratories. WHONET 5.6 software was used to analyze the distribution of pathogens and drug resistance. Results: A total of 693 strains of pathogenic bacteria were isolated, including 448 Gram-negative bacteria(64.6%), 245 Gram-positive bacteria (35.4%). The top three pathogens were 210 strains of Escherichia coli(30.3%), 87 strains of enterococcus faecium (12.6%), 76 strains of klebsiella pneumoniae (11.0%), The resistance rates of Escherichia coli to ampicillin, cefuroxime, cefazolin, ceftriaxone, piperacillin and ampicillin / sulbactam were 80.1%, 69.4%, 67.3%, 64.1%, 63.6% and 62.8%, The resistance rates of Klebsiella pneumoniae to ampicillin / sulbactam, cefuroxime and cefazolin were 65.8%, 64.5% and 61.1%, The resistance rates of Enterobacter cloacae to ceftriaxone, ceftazidime and aztreonam were 56.2%, 53.1% and 53.1%, The resistance rates of Enterococcus faecium to moxifloxacin, clindamycin, erythromycin, penicillin G, ampicillin, ciprofloxacin and levofloxacin were 100%, 90%, 76%, 72.1%, 64.4%, 64% and 62%. Conclusions: Pathogens of biliary tract infections are mainly Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae, followed by Enterococcus faecium and Enterococcus faecalis. There were many drug-resistant bacteria, so we should pay attention to bile specimen culture and drug sensitivity test.