This study was designed to demonstrate bacterial translocation following bile duct ligation and investigate preventive effects of hyperbaric oxygen on obstructive jaundice-related bacterial translocation in an animal model. Hyperbaric oxygen treatment significantly reduced the endogenous colony counts in distal ileum of normal rats both in the short (two days) and long (seven days) term. Endogenous bacteria in distal ileum significantly increased in bile duct ligated rats in the short and long term, and presence of bacterial translocation was proven by bacterial growth in mesenteric lymph nodes, liver, spleen, and blood. Short- and long-term hyperbaric oxygen treatments significantly reduced the intestinal colony counts and prevented the bacterial translocation almost completely in rats with bile duct ligation. It is concluded that obstructive jaundice causes bacterial overgrowth and translocation, and hyperbaric oxygen treatment can prevent both bacterial overgrowth and translocation effectively.