Patients with ulcerative colitis and total abdominal proctocolectomy with ileal pouch-anal anastomosis have a 50% risk of pouchitis and a 5% to 10% risk of chronic pouchitis. The goal of the study was to compare pouch microbiota and stool bile acid composition in patients with chronic pouchitis, chronic pouchitis and primary sclerosing cholangitis, and normal pouch. Patients with ulcerative colitis and ileal pouch-anal anastomosis were recruited from March 20, 2014, to August 6, 2019, and categorized into normal pouch, chronic pouchitis, and chronic pouchitis/primary sclerosing cholangitis groups. Stool samples were subjected to bile acid quantification and 16S rRNA gene sequencing. Statistical comparisons of absolute bile acid abundance and pouch microbiota α-diversity, β-diversity, and taxa abundance were performed among the patient groups. A total of 51 samples were analyzed. Both α-diversity (P = .01, species richness) and β-diversity (P = .001) significantly differed among groups. Lithocholic acid was significantly lower in patients with chronic pouchitis/primary sclerosing cholangitis than in those with chronic pouchitis (P = .01) or normal pouch (P = .03). Decreased α-diversity was associated with an increased primary to secondary bile acid ratio (P = .002), which was also associated with changes in β-diversity (P = .006). Pouch microbiota α- and β-diversity differed among patients with normal pouch, chronic pouchitis, and chronic pouchitis/primary sclerosing cholangitis. Lithocholic acid level and primary to secondary bile acid ratio were highly associated with pouch microbiota richness, structure, and composition. These findings emphasize the associations between pouch microbiota and bile acid composition in dysbiosis and altered metabolism, suggesting that secondary bile acids are decreased in chronic pouchitis.