The article presents the results of investigating 20 patients with cholelithiasis. The bile and a piece of the gallbladder were taken to analyze microbiota during a scheduled laparoscopic cholecystectomy. The study of microbiota was carried out with the cultural method and real-time PCR. Bifidobacterium spp., Bacteroides spp., Lactobacillus spp., E. coli prevailed in the taxonomic structure of isolated bacteria. Isolated Enterococcus spp. had a lot of genes encoding various factors of pathogenicity.