Background: Postoperative pancreatic fistula (POPF) is a major source of morbidity following a pancreatoduodenectomy (PD), often delaying or precluding adjuvant chemotherapy and potentially compromising long-term oncologic outcomes. While established risk models focus on anatomical and biochemical factors, the role of biliary microbiota remains underexplored. This study aimed to assess relationship between bacteriobilia and the incidence of POPF, as well as its impact on overall survival (OS) in patients undergoing a PD for pancreatic ductal adenocarcinoma (PDAC). Methods: We analyzed the medical histories of 725 patients with a pancreatic tumor who were qualified for surgery between 2017 and 2022. This retrospective cohort study included 138 patients who underwent a PD for histologically confirmed PDAC. Intraoperative bile cultures were obtained and analyzed for microbial presence and resistance patterns. Results: Bacteriobilia was detected in 76.8% of patients, including bacteria with resistance mechanisms (BRM) present in 12.3% of bile samples. Bacterial bile colonization conferred an increased odds of POPF grade B (OR 5.11; p = 0.088), whereas BRM were strongly predisposed to POPF grade C (OR 4.97; p = 0.026). Upon a multivariate analysis, bacteriobilia independently drove clinically relevant POPF and POPF grade B (OR 5.50; p = 0.034 and OR 8.04; p = 0.048, respectively), while BRM remained a key determinant of POPF grade C (OR 6.17; p = 0.047). Beyond morbidity, bile colonization markedly impaired overall survival irrespective of tumor stage (26.7 vs. 54.7 months; log-rank p = 0.009). Conclusions: Bacterial bile colonization may contribute not only to higher rates of POPF but to a significantly reduced OS in patients undergoing a PD for PDAC. Bacteriobilia should be considered as a prognostic factor for worse survival after a PD.