We focus on the ozone water to develop a novel treatment approach for staphylococci related inflammatory skin diseases. In the 95th Annual Meeting of the Japanese Pharmacological Society, we reported the anti-allergic and anti-microbial effect of 3 mg/L ozone water in a mouse model of atopic dermatitis (AD) and staphylococci, however, its effect was far from clinical application. The aim of this study is to evaluate the anti-microbial and anti-inflammatory property of high concentration (11 mg/L) of ozone water to advance our project. Ozone water showed a significant bactericidal effect against Staphylococcus aureus, S. pseudintermedius and S. lentus. Furthermore, S. pseudintermedius induced IL-1β and IL-6 secretion in human epidermal keratinocytes was significantly inhibited by pre-treatment of ozone water. In vivo experiment with a mouse model of AD, significant improvement of AD symptoms and trans epidermal water loss were also found in an ozone water treatment group. Surprisingly, local immune reactions such as type II conventional dendritic cells, effector helper T cells, and IgE-produced B cells are also regulated by ozone water application. Our findings strongly suggest that topical treatment with high concentration of ozone water significantly ameliorated AD symptoms through antibacterial effect toward staphylococci.