The gut microbiota play an essential role in protecting the host against pathogenic microorganisms by modulating immunity and regulating metabolic processes. In response to environmental factors, microbes can hugely alter their metabolism. These factors can substantially impact the host and have potential pathologic implications.  Particularly pathogenic microorganisms colonizing pancreas and biliary tract tissues may be involved in chronic inflammation and cancer evolution. To evaluate the effect of bile microbiota on survival in patients with pancreas and biliary tract disease (PBD). We investigated 152 Italian patients with cholelithiasis (CHL), cholangitis (CHA), cholangiocarcinoma (CCA), gallbladder carcinoma (GBC), pancreas head carcinoma (PHC), ampullary carcinoma (ACA), and chronic pancreatitis (CHP). Demographics, bile cultures, therapy, and survival rates were analyzed in cohorts (T death <6 months; T death <12 months; T death <18 months, T alive at 18 months). The most common bacteria in T were , and . In T, the most common bacteria were and . In T, there were no significant bacteria isolated, while in T the most common bacteria were like those found in T. and were positive predictors of survival for PHC and ACA, respectively. , and showed a high percentage of resistant bacteria to 3CGS, aminoglycosides class, and quinolone group especially at T and T in cancer patients. An unprecedented increase of in bile leads to a decrease in survival. We suggest that some strains isolated in bile samples may be considered within the group of risk factors in carcinogenesis and/or progression of hepato-biliary malignancy. A better understanding of bile microbiota in patients with PBD should lead to a multifaceted approach to rapidly detect and treat pathogens before patients enter the surgical setting in tandem with the implementation of the infection control policy.