Photodynamic therapy (PDT) has been recommended as an alternative therapy for various diseases including microbial infection. Recently, we developed a new method for the preparation of highly pure chlorin e₆ (Ce₆), which has been widely used as a second-generation photosensitizer. PDT using Ce₆ was very effective for inhibition of in vitro growth of several bacterial strains. To clarify a possibility for its clinical application, in this study, we examined in vitro and in vivo antimicrobial effects of Ce₆-mediated PDT in mice model of skin infection of Staphylococcus aureus Xen29. Inhibition zone analysis and colony forming unit (CFU) count revealed that Ce₆-mediated PDT inhibited effectively in vitro bacterial growth. In addition, biofilm formation ability of S. aureus Xen29 was decreased by Ce₆-mediated PDT. In vivo experiment, mice receiving Ce₆-mediated PDT exhibited less intensity of bioluminescent signal, showing significant inhibition of bacterial growth. Furthermore, in histopathological examination, marked neutrophilic infiltration and massive bacterial colonies were seen in control mice and mice receiving laser or Ce₆ alone, but not in mice treated with PDT. These results suggest that PDT using Ce₆ extracted by our new method can be clinically useful against bacterial infectious diseases.