There is little evidence indicating a causal linkage between bacterial translocation and postoperative infectious complication (POIC) in human studies. To investigate the correlation between the occurrence of bacterial translocation in the mesenteric lymph node (MLN) and POIC with a sensitive quantitative method using bacterium-specific ribosomal RNA (rRNA)-targeted reverse transcriptase-polymerase chain reaction (RT-qPCR). Patients who underwent major hepatectomy for biliary malignancies involving hepatic hilus were included in this study (n = 65). Mesenteric lymph nodes were harvested from the jejunal mesentery 2 times during the operation (MLN-1 harvested at laparotomy and MLN-2 harvested after tumor resection). Microorganisms were detected by a bacterium-specific rRNA-targeted RT-qPCR method. Perioperative factors and POIC were recorded prospectively. Of 65 patients, 51 completed the study. Microorganisms were detected in MLN-1 and MLN-2 in 15 (29.4%) and 19 (37.3%) patients, respectively. The detection of microorganisms in MLN-1 was significantly correlated with the incidence of preoperative cholangitis (P = 0.04), whereas the detection of microorganisms in MLN-2 was significantly correlated with the incidence of POIC (P = 0.002). In multivariate analysis, a positive result for detection of microorganisms in MLN-2 was one of the independent predictive factors of POIC (odds ratio = 26.1). Intraoperative analysis of MLNs (especially MLN-2) by rRNA-targeted RT-qPCR can strongly predict the occurrence of POIC after hepatectomy for biliary malignancy. This method is more sensitive and faster at detection of microorganisms than the conventional culture method. Therefore, we can obtain the information of bacterial translocation immediately after the surgery and can select the group of patients with high risk for POIC.