ABSTRACT: The intestinal microbiome contributes to colorectal carcinogenesis, disease progression, and response to therapy. Pathologic complete response is the therapeutic goal of neoadjuvant chemoradiation in rectal carcinoma. Nonoperative management has become an accepted strategy, and markers of complete treatment response are needed. Intestinal commensal bacteria contribute to treatment response and radiation colitis, and microbiome-targeted therapies have shown promise in clinical trials. We investigated the relationship among mucosa-associated bacteria, neoadjuvant therapy response, and radiation colitis symptoms in 57 patients who received neoadjuvant regimens with no therapy, chemotherapy only, or chemoradiation. The design was a retrospective cohort study. Microbiome profiling was performed by 16S rDNA sequencing of formalin-fixed, paraffin-embedded tissue at the proximal margin of resection. Global β diversity differed according to neoadjuvant therapy modality and was associated with radiation colitis. Taxonomic differences were detectable at phylum and lower classification levels, and radiation-induced colitis was associated with enrichment of the Bacillaceae family. Taxonomic features, including reduced Streptococcus, Lachnospiraceae, and Bacillaceae, were enriched in complete histopathologic responders to neoadjuvant therapy. Taxon-based prediction of metabolic pathways identified enrichment of prokaryotic NAD+ biosynthesis and salvage pathways in complete responders. Mucosal microbiome responses to multimodal neoadjuvant therapy reflect symptomatic radiation colitis, histopathologic evidence of radiation injury, and pathologic treatment response. Posttreatment microbiome β diversity markers of complete pathologic response may be useful in decisions to manage rectal carcinoma nonoperatively. SIGNIFICANCE: Posttreatment markers of the complete response of rectal carcinoma to neoadjuvant chemoradiation are needed to guide decisions about surgical resection. We found that mucosal microbiome β diversity, bacterial metabolic capacities, and specific taxonomic groups distinguished between complete and incomplete responders. The mucosal microbiome provides markers for complete pathologic response.