Abdominal infections including cholangitis represent a major problem in patients with perihilar cholangiocarcinoma (pCCA). Thus, we investigated bacterial colonization of the bile ducts and determined its impact on postoperative outcome focusing on abdominal infections. A cohort of 95 pCCA patients who underwent surgery between 2010 and 2019 with available intraoperative microbial bile cultures were analyzed regarding bile duct colonization and postoperative abdominal infection by group comparisons and logistic regressions. 84.2% (80/95) showed bacterial colonization of the bile ducts and 54.7% (52/95) developed postoperative abdominal infections. Enterococcus faecalis (38.8%, 31/80), Enterococcus faecium (32.5%, 26/80), Enterobacter cloacae (16.3%, 13/80) and Escherichia coli (11.3%, 9/80) were the most common bacteria colonizing the bile ducts and Enterococcus faecium (71.2%, 37/52), Enterococcus faecalis (30.8%, 16/52), Enterobacter cloacae (25.0%, 13/52) and Escherichia coli (19.2%, 10/52) the most common causes of postoperative abdominal infection. Further, reduced susceptibility to perioperative antibiotic prophylaxis (OR = 10.10, p = .007) was identified as independent predictor of postoperative abdominal infection. Bacterial colonization is common in pCCA patients and reduced susceptibility of the bacteria to the intraoperative antibiotic prophylaxis is an independent predictor of postoperative abdominal infections. Adapting antibiotic prophylaxis might therefore have the potential to improve surgical outcome pCCA patients.