Background/Aims: This study investigated the effect of perioperative synbiotics on bacterial translocation to mesenteric lymph nodes (MLNs) and occurrence of infectious complications following pancreatoduodenectomy (PD; University Hospital Medical Information Network ID 000003705). Methods: Patients who were scheduled to undergo PD were randomized to receive preoperative synbiotics or no synbiotics (control group). MLNs were harvested at laparotomy (MLN-1) and after the resection (MLN-2). Blood samples were collected before laparotomy (Blood-1) and on postoperative day 1 (Blood-2). Microorganisms in each sample were detected using a bacterium-specific ribosomal RNA-targeted reverse transcriptase-quantitative polymerase chain reaction. Results: Forty-four patients were included. In all samples, the bacterial detection rate in the MLN-1, MLN-2, Blood-1, and Blood-2 was lower in the synbiotics group than in the control group, although it did not reach a statistically significant difference. There was a significant correlation between the positivity of bacteria in the MLN-2 and Blood-2 samples (p = 0.009). The incidence rate of overall infectious complications was not significantly different between the 2 groups. Among various perioperative factors, the incidence of pancreatic fistula was the only factor that had a significant association with the incidence of infectious complications. Conclusion: The preoperative use of synbiotics did not affect the incidence of infectious complications following PD.