Rare diseases, despite their low prevalence, affect millions of individuals worldwide and are often characterized by chronic progression and a substantial impact on quality of life. In recent years, the relationship between the gut microbiota and rare diseases has emerged as an important area of research due to the growing understanding of the microbiota’s influence on host metabolism, immune responses, and neurological functions. This chapter first discusses the human microbiota, the concept of dysbiosis, and the physiological roles of microbiota-derived metabolites. It then examines the potential contribution of dysbiosis to the pathogenesis of rare diseases through biologically active molecules such as short-chain fatty acids, bile acid derivatives, and tryptophan metabolites. Selected microbiota-associated rare diseases, including Rett syndrome, Angelman syndrome, Prader–Willi syndrome, phenylketonuria, Fabry disease, Hirschsprung disease, short bowel syndrome, primary sclerosing cholangitis, cystic fibrosis, and Duchenne muscular dystrophy, are reviewed with a focus on reported alterations in microbiota composition and their possible molecular mechanisms. In addition, the therapeutic potential of microbiome-based interventions, including probiotics, prebiotics, postbiotics, fecal microbiota transplantation, and live biotherapeutic products, is discussed. Finally, the potential contributions of multi-omics technologies, artificial intelligence-assisted analyses, and personalized medicine approaches to the diagnosis and treatment of rare diseases are evaluated. Current evidence suggests that the microbiota may play a significant role in the pathogenesis of rare diseases and that microbiome-based approaches could contribute to the development of novel biomarkers and targeted therapeutic strategies in the future.