Objective: To identify whether enteric bacteria pass into the mesenteric lymph nodes (MLNs) and peritoneal cavity in calves with atresia coli and to evaluate whether the presence of bacterial translocation (BT) has an impact on the success of surgical treatment. Design: Prospective clinical study. Animals: Twenty-six client-owned calves. Interventions: During laparotomy, swab samples were collected from the peritoneal cavity and MLNs using a sterile swab stick and were submitted for microbiological analysis. Measurements and Main Results: Bacterial cultures of swab samples revealed that 65% (n = 17) of the calves experienced BT. Of these, 14 calves experienced BT to the MLNs, 9 to the peritoneal cavity, and 5 to both regions. Of the bacteria isolated from the MLNs, 72% (n = 10) were Escherichia coli. Of the samples isolated from the peritoneal fluid, 33% (n = 3) contained E. coli and 33% (n = 3) contained E. coli + coagulase-negative Staphylococcus (CNS). In calves with BT that were discharged (n = 13) and without BT that were discharged (n = 7), the median survival was 30 days; these data were found to be similar in the 2 groups. Conclusions: This study revealed that BT is observed in the majority of atresia coli cases. E. coli is more common in BT, and translocation occurs primarily through the lymphatic route. These results suggest that the presence of BT is closely related to the success of the operation for correction of atresia coli.