Dysbiosis and pathobionts contribute to inflammation and the risk of colitis-associated carcinoma (CAC) in animal models, but their roles in humans with this uncommon disease are unknown. We identified microbiome differences in human CAC compared with longstanding inflammatory bowel disease (IBD) and sporadic colorectal carcinoma (CRC). Twenty-four CAC resections were matched with CRC and IBD controls. Methods included histopathology, 16S rDNA metagenomics, and pathobiont-specific qPCR. Beta diversity differed by diagnosis (PERMANOVA p = 0.007). The distinguishing taxa included Akkermansia enriched in CRC, and Bacteroides spp. enriched in IBD. The non-neoplastic mucosae presented distinct beta diversity (p = 0.005), but the CAC/CRC tumor microbiomes were similar (p = 0.7). Within metastases and margins, Enterobacteriaceae were enriched in CAC, and Bacteroidales in CRC. Pathobiont-specific qPCR confirmed a greater frequency of pks+ E. coli and enterotoxigenic Bacteroides fragilis in CAC than IBD. High alpha diversity was associated with active inflammation, advanced cancer stage, and shorter overall survival (log-rank p = 0.008). Mucosal microbiomes distinguish CAC from longstanding IBD, implicating pathobionts as markers for disease progression. Integrating our findings with prior animal model research, pathobionts promote carcinogenesis in IBD patients through genotoxicity and host cell signaling.