Recent studies have revealed that oral, gut, and intratumoral microbial dysbiosis significantly affects tumor progression, therapy resistance, and prognosis in cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC) patients. However, the biliary microbiome, which directly interacts with malignant tissues, remains poorly understood. In this study, we analyzed the bile microbiota from 17 CCA, 15 PDAC, and 40 choledocholithiasis (CDL) patients using bacterial 16 S rRNA and fungal ITS sequencing. Principal coordinate analysis revealed significant differences in microbial communities between the cancer and CDL groups. The microbial community structure in each group demonstrated a specific pattern. Linear discriminant analysis revealed Streptococcus, Sphingomonas, and Bacillus enrichment in CCA patients, Neisseria, Sphingomonas, and Caulobacter in PDAC patients were more prevalent compared with CDL patients. Caulobacter was more prevalent, wheares Campylobacter was less in PDAC patients than in CCA patients. Fungal DNA was detected in ~ 50% of the samples, with CCA and PDAC patients. KEGG pathway analysis revealed altered metabolic pathways, including peptidoglycan, sphingolipid, and fatty acid metabolism and bile acid metabolism, in CCA and PDAC patients. These findings highlight the potential role of the biliary microbiome in CCA and PDAC pathogenesis, offering new insights into disease mechanisms and biomarkers.