Background Metabolic syndrome and obesity occur commonly in long-term pediatric cancer survivors and exacerbate other chronic conditions. The intestinal microbiome is associated with metabolic syndrome and obesity in the general population, and is perturbed during cancer therapy. We aimed to determine if long-term survivors of pediatric cancer would have reduced bacterial microbiome diversity, and if these findings would be associated with components of the metabolic syndrome, obesity, and chronic inflammation. Methods We performed a cross-sectional study examining the intestinal microbiome, clinical factors, and biomarkers between 35 long-term survivors and 32 age, sex, and race matched controls. All subjects were ages 10-40, and survivors were at least five years from the time of diagnosis. Results Survivors had decreased alpha diversity compared to controls (Shannon index p=0.001, Simpson index p=0.032) and differently abundant bacterial taxa. Further, among survivors, those who received radiation to the central nervous system or abdomen/pelvis had decreased alpha diversity compared to those that did not receive radiation (Shannon and Simpson p<0.05 for both). Although, no specific component of metabolic syndrome or cytokine was associated with measures of alpha diversity, survivors with low adiponectin-lectin ratio, elevated body mass index, and elevated C-Reactive protein had differently abundant taxa compared to those with normal measures. Conclusions The microbiome of cancer survivors remains less diverse than controls even many years after diagnosis, and exposure to radiation may lead to further loss of diversity in survivors. The microbiome may be associated with metabolic syndrome and chronic inflammation in survivors.