Pelvic radiotherapy is known to evoke intestinal mucositis and dysbiosis. Currently, there are no effective therapies available to mitigate these injuries, which is partly due to a lack of insight into the events causing mucositis and dysbiosis. Here, the complex interplay between the murine host and its microbiome following pelvic irradiation was mapped by characterizing intestinal mucositis along with extensive 16S microbial profiling. We demonstrated important morphological and inflammatory implications within one day after exposure, thereby impairing intestinal functionality and inducing translocation of intraluminal bacteria into mesenteric lymph nodes as innovatively quantified by flow cytometry. Concurrent 16S microbial profiling revealed a delayed impact of pelvic irradiation on beta diversity. Analysis of composition of microbiomes identified biomarkers for pelvic irradiation. Among them, members of the families Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae were differentially affected. Altogether, our unprecedented findings showed how pelvic irradiation evoked structural and functional changes in the intestine, which secondarily resulted in a microbiome shift. Therefore, the presented in vivo irradiation-gut-microbiome platform allows further research into the pathobiology of pelvic irradiation-induced intestinal mucositis and resultant dysbiosis, as well as the exploration of mitigating treatments including drugs and food supplements.