There are no data evaluating the microbiome in congenital heart disease following cardiopulmonary bypass. The authors evaluated patients with congenital heart disease undergoing cardiopulmonary bypass and noncardiac patients undergoing surgery without bypass. Patients with congenital heart disease had differences in baseline microbiome compared with control subjects, and this was exacerbated following surgery with bypass. Markers of barrier dysfunction were similar for both groups at baseline, and surgery with bypass induced significant intestinal barrier dysfunction compared with control subjects. This study offers novel evidence of alterations of the microbiome in congenital heart disease and exacerbation along with intestinal barrier dysfunction following cardiopulmonary bypass.