Radiotherapy is a cornerstone of cancer treatment. Initially, research concentrated on the intrinsic characteristics of cancer cells in determining radiosensitivity. However, recent findings underscore the pivotal role of both the tumor microenvironment (TME) and the host microbiota in modulating the response to radiation. The human microbiota, comprising microorganisms that reside within the body, impacts the efficacy of radiotherapy both locally through tumor-infiltrating microbes (TiMs) and systemically, primarily via the gut microbiota. The microbiota modulates radiation response through diverse mechanisms, including immune modulation, regulation of hypoxia, reactivation of oncovirus, induction of immune exhaustion, and activation of TME. Dysbiosis may contribute to increased radioresistance by hindering anti-tumor immune reactivation, exacerbating tumor hypoxia, and fostering an immunosuppressive TME. Conversely, radiotherapy can directly impact microbial composition, which might affect therapeutic efficacy. This chapter offers a comprehensive overview of both experimental and clinical evidence regarding microbiota’s influence on radiation response, elucidating potential mechanisms and exploring microbiome-targeted strategies to enhance radiotherapy efficacy. Particular emphasis is placed on the role of probiotics as potential radiosensitizers. Elucidating these interactions presents a promising opportunity to develop Personalized Radiotherapy approaches, incorporating microbiota profiling to optimize cancer treatment outcomes while reducing adverse effects.