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Background: Oral mucositis is probably the most debilitating complication that can arise in treating a patient with head and neck cancer. Little is known about the impacts of oral microbiota on the initiation and progression of mucositis. Methods: Based on 16S rRNA gene sequencing, dynamic changes in oral bacterial profile as well as correlations between the severity of mucositis and bacterial shifts during radiotherapy were investigated. Findings: Our results revealed that bacterial community structure altered progressively during radiation therapy, in parallel with a marked increase in the relative abundance of some Gram-negative bacteria. Patients who eventually developed severe mucositis harbored a significantly lower bacterial alpha diversity and higher abundance of Actinobacillus during the phase of erythema – patchy mucositis. Accordingly, a random forest model for predicting exacerbation of mucositis was generated, which achieved a high predictive accuracy (AUC) of 0.89. Interpretation: Oral microbiota changes correlate with the progression and aggravation of radiotherapy-induced mucositis in patients with nasopharyngeal carcinoma. Microbiota-based strategies can be used for the early prediction and prevention of the incidence of severe mucositis during radiotherapy.