Background and Aims: Enterococcus has emerged as a virulent species; Enterococcus faecium especially has arisen as a source of nosocomial infections. Furthermore, specific Enterococcus faecalis species are significantly associated with anastomotic leakage in rodent studies. The objective of this study was to investigate whether the occurrence of Enterococci ( E. faecium and E. faecalis) obtained from drain samples was associated with leakage in humans undergoing pancreaticoduodenectomy. Materials and Methods: All patients undergoing pancreaticoduodenectomy had a peritoneal drain sample sent for culturing between postoperative days 3 and 10. Postoperative pancreatic fistulas were defined and classified according to the International Study Group of Pancreatic Fistula. Bile leakage was radiologically verified. Postoperative complications were classified according to the Dindo–Clavien classification. Results: A total of 70 patients were eligible and enrolled in this study. Anastomosis leakage was observed in 19 patients; 1 leakage corresponding to the hepaticojejunostomy and 18 pancreatic fistulas were identified. In total, 10 patients (53%) with leakage had Enterococci-positive drain samples versus 12 patients (24%) without leakage [odds ratio (OR) = 5.1, 95% confidence interval (CI) = 1.4-19.4, p = 0.02]. Preoperative biliary drainage with either endoscopic stenting or a percutaneous transhepatic cholangiography catheter was associated with the occurrence of Enterococci in drain samples (OR = 5.67, 95% CI = 1.8-12.9, p = 0.003), but preoperative biliary drainage was not associated with leakage (OR = 0.45, 95% CI = 0.1-1.7, p = 0.23). Conclusion: Enterococci in drain sample cultures in patients undergoing pancreaticoduodenectomy occurs significantly more among patients with anastomotic leakage compared to patients without leakage.