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The human microflora is a complex ecosystem composed of diverse microorganisms mainly distributed in the epidermal and mucosal habitats of the entire body, including the mouth, lung, intestines, skin, and vagina. These microbial communities are involved in many essential functions, such as metabolism, immunity, host nutrition, and diseases. Recent studies have focused on the microbiota associated with cancers, particularly the oral and intestinal microbiota. Radiotherapy, the most effective cytotoxic modality available for solid tumors, contributes to the treatment of cancer patients. Mounting evidence supports that the microbiota plays pivotal roles in the efficacy and prognosis of tumor radiotherapy. Here, we review current research on the microbiota and cancer development, and describe knowledge gaps in the study of radiotherapy and the microbiota. Better understanding of the effects of the microbiome in tumorigenesis and radiotherapy will shed light on future novel prevention and treatment strategies based on modulating the microbiome in cancer patients.