One of the main causes of cancer-related death globally is colorectal cancer (CRC). Emerging research highlights the gut microbiota’s important involvement in CRC aetiology, underscoring the complex interplay between host genetics, microbial dysbiosis, and tumour development. This chapter delves into the fundamental aspects of gut microbiota in CRC, exploring how microbial imbalances can promote or suppress tumorigenesis through mechanisms like inflammation, immune modulation, and production of carcinogenic metabolites. In addition to examining key microbial species implicated in CRC, such as Fusobacterium nucleatum and Bacteroides fragilis, the chapter outlines the most widely used analytical techniques for studying gut microbiota. These techniques include 16S ribosomal ribonucleic acid (rRNA) sequencing and metabolomics, which have enabled the identification of microbial signatures associated with cancer progression. Despite the advances in microbiome research, significant limitations persist. Furthermore, the lack of standardized protocols for microbiota analysis complicates reproducibility across studies. This chapter highlights these limitations and calls for the development of more refined techniques and integrative approaches that combine microbiome analysis with other omics technologies. In summary, comprehending the function of gut microbiota in colorectal cancer (CRC) is crucial for creating innovative approaches for diagnosis, prognosis, and treatment. This chapter delves into a detailed overview of the current knowledge, methodologies, and challenges in gut microbiota research in the context of colorectal cancer.