The present study aimed to evaluate in vitro and in vivo antibacterial activity of hydroxyl radical generation system by photolysis of H2O2, which is a new disinfection system for the treatment of oral infection diseases such as periodontitis developed in our laboratory. Firstly, generation of the hydroxyl radical by the photolysis of H2O2 in which 1 mol I-1 H2O2 was irradiated with a dual wavelength-light emitting diode (LED) at wavelengths of 400 and 465 nm was confirmed by applying an electron spin resonance-spin trapping technique. Secondly, the bactericidal effect of the system was examined under a similar condition in which Staphylococcus aureus suspended in 1 mol I-1 H2O2 was irradiated with LED light, resulting in substantial reduction of the colony forming unit (CFU) of the bacteria within a short time as 2 min. Finally, in vivo antibacterial effect of the photolysis of H2O2 on a rat model of S. aureus infection was evaluated by a culture study. Since a significant reduction of recovered CFU of S. aureus was obtained, it is expected that in vitro antibacterial effect attributable to hydroxyl radicals generated by photolysis of H2O2 could be well reflected in in vivo superficial bacterial infection. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.