Objective To explore the pathogen distribution and the characteristics of antibiotics use of patients with positive bile culture in order to provide evidence for appropriate antibiotic use. Methods Using a patient-based approach, the clinical and laboratory data of patients with positive bile culture between December 1st 2016 and November 30th 2017 were retrospectively analyzed. The pathogen distribution and antibiotics use of patients with bililary duct infections and colonizations were analyzed. Multidrug-resistant organism infections of patients with bililary duct infections were studied. Results There were 299 submitted bililary samples and in which 158 were culture-positive (52.8%). One hundred and ten strains of pathogens were found in 79 patients with positive bile culture, including 66 strains of Gram-negative (G–) organisms (60.0%), 37 strains of Gram-positive (G+) organisms (33.6%), and 7 strains of fungi (6.4%). The top three G– organisms were Escherichia coli (25 strains, 22.7%), Klebsiella pneumoniae (9 strains, 8.2%), and Acinetobacter baumanii (7 strains, 6.4%). The top three G+ organisms were Enterococcus faecium (10 strains, 9.1%), Enterococcus faecalis (6 strains, 5.5%), and coagulase negativeStaphylococcus (6 strains, 5.5%). The number of patients with bililary duct infections and colonizations were 42 and 37, respectively, with pathogens occupied mainly by G– bacteria. Ten strains of multidrug-resistant organisms were isolated from patients with bililary duct infections. Compared to patients with non-multidrug-resistant organism infections, the length of antibiotics use was longer in patients with multidrug- resistant organism infections (t=2.129, P=0.039). The rate of target therapy for antibiotics in patients with bililary duct infections was 76.2%. The rate of proper antibiotics use was 16.2% before positive bile culture and 78.4% after positive bile culture in patients with bililary duct colonizations. Conclusions Pathogens isolated from bile culture of infection and colonization are predominantly G– organisms. The bile culture and blood culture should be done for patients with suspected bililary duct infection. Infection and colonization should be distinguished for positive bile culture and antibiotic should be chosen according to drug susceptibility test results.