BACKGROUND A functional interplay between BAs and microbial composition in gut is a well-documented phenomenon. In bile, this phenomenon is far less studied, and with this report, we describe the interactions between the BAs and microbiota in this complex biological matrix. Methodology. Thirty-seven gallstone disease patients of which twenty-one with Opisthorchis felineus infection were enrolled in the study. The bile samples were obtained during laparoscopic cholecystectomy for gallstone disease operative treatment. Common bile acid composition was measured by LC-MS/MS. Gallbladder microbiota were previously analyzed with 16S rRNA gene sequencing on Illumina MiSeq platform. The associations between bile acid composition and microbiota were analyzed. RESULTS Bile acid signature and Opisthorchis felineus infection status exert influence on beta-diversity of bile microbial community. Direct correlations were found between taurocholic acid, taurochenodeoxycholic acid concentrations, and alpha-diversity of bile microbiota. Taurocholic acid and taurochenodeoxycholic acid both show positive associations with the presence of Chitinophagaceae family, Microbacterium and Lutibacterium genera, and Prevotella intermedia. Also, direct associations were identified for taurocholic acid concentration and the presence of Actinomycetales and Bacteroidales orders, Lautropia genus, Jeotgalicoccus psychrophilus, and Haemophilus parainfluenzae as well as for taurochenodeoxycholic acid and Acetobacteraceae family and Sphingomonas genus. There were no differences in bile acid concentrations between O. felineus-infected and noninfected patients. Conclusions/Significance. Associations between diversity, taxonomic profile of bile microbiota, and bile acid levels were evidenced in patients with cholelithiasis. Increase of taurochenodeoxycholic acid and taurocholic acid concentration correlates with bile microbiota alpha-diversity and appearance of opportunistic pathogens.