ObjectiveTo investigate isolation of pathogens from bile and clinical characteristics of patients with hepatobiliary diseases. MethodsBacterial culture result of bile and related clinical data of patients with hepatobiliary diseases in a hospital were collected and analyzed by retrospective survey. ResultsA total of 406 bile specimens from patients with hepatobiliary diseases were collected, the positive rate of culture was 64.53%. Of 262 positive specimens, 62.21%（n=163）, 32.83%（n=86）, and 4.96%（n=13） were isolated single pathogen, 2 kinds of pathogens，and 3 kinds of pathogens respectively. 374 pathogenic strains were isolated, 242（64.71%）, 131 （35.02%）, and 1（0.27%）were gramnegative bacteria, grampositive bacteria, and fungus respectively. Patients with cirrhosis of liver, history of hepatobiliary surgery, and cholelithiasis had higher isolation rates of pathogens from bile than control group(all P<0.05), isolation rates of pathogens from bile in patients with cholelithiasis of different sites were varied; but there was no significant differences among patients of different age, gender, and whether or not with hepatobiliary tumors(all P>0.05). There were no statistical difference in constitute of pathogenic species from bile between patients with and without gallstones, as well as with and without history of hepatobiliary surgery（both P＞0.05）；while constitute of pathogenic species from bile between patients with and without cirrhosis of liver was statistically different（χ2=14.058，P=0.001）. ConclusionPathogens from bile in patients with hepatobiliary diseases are mainly Enterobacteriaceae and Enterococcus spp. which caused single infection. Positive culture rate of pathogens from bile is higher in patients with cholelithiasis, history of hepatobiliary surgery, and liver cirrhosis.