Colorectal cancer (CRC) is a prevalent global malignancy with complex etiologies, including microbiota alterations. This study investigates gut microbiota and biofilm-producing bacteria in 35 Thai CRC patients, analyzing paired normal and tumor biopsy samples. Bacterial DNA from the V3-V4 region of 16S rRNA was sequenced, and biofilms were visualized via scanning electron microscopy and fluorescence in situ hybridization (FISH). Results revealed Firmicutes as the dominant phylum, followed by Bacteroidota, Proteobacteria, and Fusobacteriota, with Fusobacteriota and Bacteroidota notably enriched in left-sided CRC. Key biofilm producers-Bacteroides fragilis, Fusobacterium nucleatum, and Pasteurella stomatis-showed significantly higher gene expression in tumor tissues. Dense biofilms and higher Fusobacterium abundance, localized within the crypts of Lieberkuhn, were observed in CRC tissues. These findings highlight CRC-associated microbiota alterations and pathogenic biofilm production, emphasizing a spatial relationship between tumor location and microbial distribution, with potential implications for understanding CRC pathogenesis and therapeutic targeting.