AbstractStaphylococcus aureus(S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the ‘superbug’Methicillin‐resistant S. aureus(MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin‐1E‐OG9, isolated from the skin secretions ofOdorrana grahami, which shows potent antibacterial activity, especially againstS. aureus.Based on the characteristics of the ‘Rana Box’, we designed a set of brevinin‐1E‐OG9 analogues to explore its structure–activity relationship. Brevinin‐1E‐OG9c‐De‐NH2exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat‐killed microbes. As a result, brevinin‐1E‐OG9c‐De‐NH2might represent a promising candidate for the treatment ofS. aureusskin infections.