Radiotherapy has an established role in the clinical treatment of patients with a variety of cancers owing to the ability to preferentially kill malignant cells mostly while sparing their non-malignant counterparts. Results from phase I-II trials also suggest that radiotherapy can have therapeutically relevant immunostimulatory effects, especially when combined with immune-checkpoint inhibitors. Over the past two decades, evidence has emerged showing that intestinal microbial communities have a major influence on the immunological tonus of patients with cancer and can influence sensitivity to various immunotherapies, including immune-checkpoint inhibitors and chimeric antigen receptor T cells. Here, we critically discuss the effects of such microbial ecosystems on radiotherapy-induced toxicities and tumour-targeting immune responses, with a focus on the clinical potential of these relationships for predictive and therapeutic clinical applications.