Background and Aims Biliary tract infection is a common acute abdominal disease. It can lead to septic shock and even death if not treated on time. So, knowledge of the bacterial species and their antimicrobial sensitivity patterns are critically important for guiding clinical treatment strategies. This study was conducted to analyze the distribution and drug resistance of pathogenic bacteria in bile culture obtained by endoscopic retrograde cholangiopancreatography (ERCP) in patients with biliary tract diseases, so as to provide basis for rational use of antibiotics in patients with biliary tract infection.Methods The data of 1 141 patients undergoing ERCP for biliary tract disease with bile culture and drug susceptibility test in Foshan First People's Hospital from January 2016 to December 2019 were retrospectively analyzed. The identification of microbial strains isolated from the bile samples of the patients and drug susceptibility test were determined by VITEK2-COMPACT automatic microbial identification system. Further, the results between patients with gallstones and those without gallstones were analyzed and compared.Results A total of 745 strains of pathogens were isolated from bile culture, including 488 types of gram-negative bacteria (65.5%), 195 types of gram-positive bacteria (26.2%) and 62 types of fungi (8.3%). The top three pathogens were Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis, accounting for 33.4%, 12.6% and 8.2%, respectively. The Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis showed the highest resistance rate to trimethoprim, ampicillin and clindamycin, respectively. The antibiotic resistance patterns of Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis to different antibiotics varied from 2016 to 2019. The incidence of biliary tract bacterial infection in patients with gallstones was higher than that in patients without gallstones (67.5% vs. 58.8%), but patients without gallstones were more likely to develop antibiotic resistance.Conclusion Both the bacterial species composition and their antibiotic resistance pattern of the pathogens of biliary infection are dynamically changed over time. Clinical treatment should be directed by bile culture and sensitivity test results, so as to ensure rational use of antibiotics, maximizing the benefit of patients and reducing the generation of drug-resistant strains.