This volume presents a collection of both classic and modern experimental methods designed to highlight the biology, pathogenicity, and host interactions of Fusobacterium nucleatum. The chapters in this book provide practical guidance spanning microbiology, molecular biology, imaging, genetic engineering, metabolomics, and in vivo modeling. Some of the topics discussed in this volume are methods for visualizing and isolating F. nucleatum within its native oral environment; essential cellular processes such as surface protein assembly and peptidoglycan biosynthesis; protocols for asymmetric coculture systems; 3D tumor spheroid models; untargeted metabolite profiling of infected mammalian cells; and several in vivo platforms – from murine models of periodontitis, oral cancer, and CRC to a Caenorhabditis elegans infection model. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Fusobacterium nucleatum: Methods and Protocols is a fundamental resource for both new and experienced researchers looking to investigate F. nucleatum virulence mechanisms, colonization strategies, and its increasing contributions to diseases such as CRC, oral squamous cell carcinoma, and periodontitis.