Gallstone disease (GSD) has, for many years, remained a high-cost, socially significant public health problem. Over the past decade, a number of studies have been carried out—both in humans and in animal models—confirming the role of the microbiota in various sections of the gastrointestinal tract as a new link in the etiopathogenesis of GSD. The microbiome of bile correlates with the bacterial composition of saliva, and the microbiome of the biliary tract has a high similarity with the microbiota of the duodenum. Pathogenic microflora of the oral cavity, through mechanisms of immunomodulation, can affect the motility of the gallbladder and the expression of mucin genes (MUC1, Muc3, MUC4), and represent one of the promoters of stone formation in the gallbladder. The presence of H. pylori infection contributes to the formation of gallstones and affects the occurrence of complications of GSD, including acute and chronic cholecystitis, cholangitis, pancreatitis. Intestinal bacteria (Clostridium, Bifidobacterium, Peptostreptococcus, Bacteroides, Eubacterium, and Escherichia coli) participating in the oxidation and epimerization of bile acids can disrupt enterohepatic circulation and lead to the formation of gallstones. At the same time, cholecystectomy due to GSD leads to the further transformation of the composition of the microbiota in various parts of the gastrointestinal tract, increasing the risk of developing stomach cancer and colorectal cancer. Further research is required to determine the possibility of using the evaluation of the composition of the microbiota of the gastrointestinal and biliary tracts as an early diagnostic marker of various gastroenterological diseases.