An increasing amount of evidence suggests the emerging role of the gut microbiota in the development of colorectal cancer (CRC). This study aimed to elucidate the architecture of microbial communities within normal and neoplastic colonic mucosa. Methods: Microbiota were analyzed by NGS and by an ensemble of metagenomics analysis tools in a total of 69 tissues from 9 patients with synchronous colorectal neoplasia and adenomas (27 specimens: 9 from normal tissues, 9 adenomas, and 9 tumours), 16 patients with only colonic adenomas (32 specimens: 16 from normal tissues and 16 adenomas), and from healthy subjects (10 specimens of normal mucosa). Results: Weak differences were observed in alpha and beta metrics among the synchronous tissues from CRC and controls. Through pairwise differential abundance analyses of sample groups, an increasing trend ofRikenellaceae, PseudomonasandFusobacterium,and decreasing trends ofStaphylococcus,ActinobacillusandGemmigerwere observed in CRC, whileStaphylococcusandBifidobacteriumwere decreased in patients with only adenomas. At RT-qPCR analysis,Fusobacterium nucleatumwas significantly enriched in all the tissues of subjects with synchronous colorectal neoplasia. Conclusion: Our findings provide a comprehensive view of the human mucosa-associated gut microbiota, emphasizing global microbial diversity mostly in synchronous lesions and proving the constant presence ofFusobacterium nucleatum,with its ability to drive carcinogenesis.