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Preoperative biliary drainage (PBD) through endoscopic retrograde cholangiopancreatography (ERCP) is advocated to reduce complications following pancreaticoduodenectomy. However, randomized trials have provided inconclusive results. This study aimed to investigate the microflora colonizing preoperatively placed biliary stents in patients with neoplasms and their resistance to antibiotic treatment. Data from 313 patients who underwent pancreaticoduodenectomy for neoplasms between 2010 and 2022 were analyzed. Among them, 171 patients underwent preoperative stent drainage, with 115 stents subjected to microbial culture. The stents were predominantly plastic. Microbial analysis revealed that 85.2% of the stents were positive for pathogens, with Gram-negative bacteria being the most prevalent (63%). Fungi were isolated in 37.4% of cultures. Klebsiella pneumoniae exhibited multidrug resistance in 69.4% of cases, and pan-resistant Pseudomonas aeruginosa was found in three cultures. The study found a significant association between waiting time to surgery and positive stent cultures. The presence of a stent increased the risk of changes in the microbiome. Interestingly, the presence of anaerobic strains was not detected in the sample. These findings suggest that biliary stents harbor a diverse range of microflora, which may have potential implications for postoperative outcomes, although direct clinical outcomes were not assessed in this study.