BackgroundEmerging evidence highlights the role of biliary microbiota in hepato-biliary-pancreatic diseases. The characteristics of biliary microbiota in malignant bile duct obstruction remain poorly understood. This study aims to investigate the composition and metabolic functions of biliary microbiota in patients with malignant obstruction.MethodsEligible patients were enrolled in this prospective study at First Affiliated Hospital of Soochow University between December 2022 and October 2023, including distal cholangiocarcinoma, hilar cholangiocarcinoma, pancreatic ductal adenocarcinoma, periampullary carcinoma, and gallbladder carcinoma. The patients with choledocholithiasis served as controls. Bile samples were collected via endoscopic retrograde cholangiopancreatography. Microbiota identification was performed using 16S rRNA sequencing, while bile acids were analyzed using mass spectrometry.ResultsA total of 56 patients were successfully enrolled in this study, 25 in the tumor group and 31 in the stone group. A distinct biliary microbial community was observed in patients with malignant bile duct obstruction, consisting of Prevotella, Uruburuella, Atopostipes, Clostridium IV, Halomonas, Tannerella, Porphyromonas, Achromobacter, Rouxiella, Campylobacter, Corynebacterium, Turicibacter, Muribaculum, Selenomonas, and Alloprevotella at genus level. Notably, Clostridium IV, Halomonas, Rouxiella, and Turicibacter were exclusively present in the tumor group. Bile acid levels were significantly lower in the tumor group (P < 0.05), except for ursodeoxycholic acid and taurocholic acid. Additionally, 22 metabolic pathways were enriched in the tumor group.ConclusionsThis study elucidates the community and metabolic potential of biliary microbiota in malignant bile duct obstruction. The findings offer valuable insights for disease assessment and provide a foundation for further research into the role of biliary microbiota in malignancy.