OBJECTIVES: An antimicrobial technique utilizing hydroxyl radicals generated by the photolysis of 3% H2O2 has been developed recently. The present study aimed to evaluate the effect of H2O2 photolysis treatment on tooth demineralization caused by Streptococcus mutans biofilm. MATERIALS AND METHODS: To induce tooth demineralization, S. mutans biofilm was allowed to form on the maxillary first molars collected from Wistar rats via 24-h culturing. The samples were immersed in 3% H2O2 and irradiated with 365-nm LED (H2O2 photolysis treatment). Viable bacterial counts in the biofilm were evaluated immediately after treatment and after an additional 30-h culturing by colony counting. The acidogenicity of the biofilm, re-established 30 h after treatment, was assessed by measuring the pH. The effect of H2O2 photolysis treatment on tooth demineralization was assessed by measuring the depth of the radiolucent layer in micro-CT images. RESULTS: H2O2 photolysis significantly reduced viable bacterial counts in the biofilm to 3.7 log colony forming units (CFU)/sample, while the untreated group had 7.9 log CFU/sample. The pH of the biofilm re-established after treatment (6.6) was higher than that of the untreated group (5.3). In line with the pH measurement, the treatment group had a significantly lower depth of radiolucent layer in dentin than the untreated group. CONCLUSIONS: H2O2 photolysis treatment was effective not only in killing the biofilm-forming S. mutans but also in lowering the acidogenicity of the biofilm. Thus, this technique could inhibit tooth demineralization. CLINICAL RELEVANCE: H2O2 photolysis can be applicable as a new dental caries treatment.