Radiotherapy is a common treatment for head and neck cancer but often causes oral mucositis, which reduces quality of life. Recent studies suggest that radiotherapy affects the oral microbiota, but whether it contributes to the severity of mucositis has been unclear. This study investigated the association between radiotherapy-induced changes in the oral microbiota and the severity of mucositis using 16S rRNA gene sequencing. Oral samples were collected before, during, and after radiotherapy and were analyzed for changes in bacterial composition and diversity using the Shannon index and Chao1 index. Severity of mucositis was assessed, and its association with changes in bacterial groups was investigated. In total, 43 patients participated in the study.