A recently developed antimicrobial technique utilizing hydroxyl radicals generated by hydrogen peroxide (H2O2) photolysis represents a promising new therapy for preventing and treating dental caries. The present study compared the antimicrobial time-kill kinetics of H2O2 photolysis, conventional antiseptics, and antimicrobial photodynamic therapy (aPDT) against biofilm-forming Streptococcus mutans (cariogenic bacteria) grown on hydroxyapatite disks. H2O2 photolysis was performed by irradiating the biofilm immersed in 3% H2O2 with 365-nm light-emitting diode (LED) light at an irradiance of 1000 mW/cm(2) for up to 1.5 min. Antiseptic treatments consisted of 0.2% chlorhexidine gluconate, 0.5% povidone-iodine, and 3% H2O2. The biofilm was immersed in each antiseptic for up to 4 min. aPDT was performed by irradiating the biofilm immersed in 100 Oil methylene blue or toluidine blue O with 655-nm laser light at 1000 mW/cm(2) for up to 4 min. Based on the time kill assay, the decimal reduction value (D-value) of each treatment was determined. With a D-value of 0.06 min, H2O2 photolysis exhibited the highest bactericidal effect against biofilm-forming S. mutans. In contrast, antiseptics and aPDT exerted a slower bactericidal effect, with D-values of 0.9-2.7 min. In conclusion, the antimicrobial technique based on H2O2 photolysis using 365-nm LED represents a strong adjunctive chemotherapy for dental caries treatment.