Involvement of bacteria in the pathogenesis of biliary tract is largely unknown. In this study, we investigated differences in the microbiota of biliary tissue among adult patients with choledocholithiasis during endoscopic retrograde cholangiography (ERCP). 16S rDNA sequencing of bile samples, culture, and data of the medication history, underlying diseases and liver function tests were used for interpretation of differences observed in composition of bacterial taxa detected in the patients. The most common phyla found among the bile samples were included Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria/Chloroplast, Fusobacteria, Acidobacteria, and Deinococcus-Thermus. Infection with anaerobic and microaerophilic bacteria showed host specificity, where Fusobacter, Prevotella, Veillonella, Propionibacter, Gemella, and Helicobacter coexist in same patients. Coexistence of anaerobic bacteria was mainly limited to patients with pancreatic cancer. Clostridium and Peptoclostridium spp. were detected in 80% and 86% of the patients, where highest relative abundance rates were detected in patients with elevated ALP levels and leukocytosis, respectively. Patients with leukocytosis presented higher rates of colonization with Pseudomonas, Cronobacter, Enterococcus, Klebsiella, Peptoclostridium, and Streptocossus. H. pylori was detected in 4 patients with and 1 patient without CBD stone. Higher diversity in the bacterial population was detected in patients with CBD stone compared with those lacking the stone, as richness of an unclassified member of Alphaproteobacteria, plus Helicobacter, Enterobacter/Cronobacter spp., Sphingomonas, Prevotella, Fusobacterium and Aeromonas were detected. Further studies are needed to show impact of these bacteria in human health or diseases through their interaction with host and virulence potential.