The presence of positive bile culture during intraoperative procedures has been associated with elevated morbidity and mortality rates in hepatobiliopancreatic surgeries, contributing to increased healthcare expenditures. However, the precise impact of bactobilia on the development of postoperative complications remains uncertain due to existing disparities in the published literature. In this retrospective cohort study, we assessed 137 patients who underwent major hepatobiliopancreatic surgery to examine the relationship between intraoperative bile culture outcomes and subsequent postoperative infectious complications. Among patients with bactobilia, a significant 35.1% exhibited systemic or local infectious complications, whereas only 11.1% of those with negative culture results experienced any infectious complications (p=0.002). Similarly, a notable difference was observed in the incidence of surgical site infections, with 24.3% in the bactobilia group compared to 7.9% in the negative culture group (p=0.01). A total of 74 monomicrobial cultures with microbiological growth were isolated, predominantly featuring Gram-negative microorganisms, primarily Enterobacteriaceae in 49 cultures. Escherichia coli was identified in 37.8% of positive cultures, while Klebsiella pneumoniae was evident in 21.6%. Gram-positive microorganisms were present in 10 cultures, with Enterococcus emerging as the prevailing species. The logistic regression model identified a positive bile culture as an independent factor significantly associated with infection development (OR: 2.26; 95% confidence interval: 1.23-11; p=0.02). Considering the limitations of the study, these findings underscore the critical importance of conducting bile cultures during the intraoperative phase to enable vigilant monitoring and prompt management of infectious complications.