Orobol, a metabolite of genistein, is rare in natural soybean. Several studies have revealed the immune-controlling effects of orobol on inflammatory diseases. Furthermore, a few studies have demonstrated that orobol decreases pro-inflammatory compounds resulting in the alleviation of allergic reactions. However, the relationship between orobol and atopic dermatitis (AD) in animal models has not been revealed. Therefore, we sought to investigate the effects of orobol on AD-like symptoms. AD-like symptoms and skin lesions were induced by repeated topical application of Dermatophagoides farinae extract (DFE) on the skin of NC/Nga mice. Topical application of orobol attenuated DFE-induced AD-like symptoms and transepidermal water loss and increased skin hydration. Histopathological analysis revealed that orobol alleviated DFE-induced eosinophil and mast cell infiltration into the skin. These observations occurred concomitantly with the downregulation of inflammatory markers including serum TARC, MDC, and IgE. In addition, orobol alleviated dorsal Th2 cytokines such as IL-4 and IL-13. Pre-treatment of orobol decreased the activity of the MAPKs and NF-κB signalling cascade in the TNFα/IFNγ-induced HaCaT cell line. These results suggest that orobol, a natural dietary isoflavone, has therapeutic efficacy for the prevention and treatment of AD.