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This study aimed to investigate the skin irritancy with unripe Citrus grandis Osbeck extract and to determine the effect of cotton underclothes dyed with the citrus extract on the severity of atopic dermatitis by in-vivo methodology. The skin irritancy from the extract was assessed by Kawai’s method and the severity of atopic dermatitis was evaluated by measuring the quantitative index of SCORAD for human subjects wearing the citrus-dyed underclothes. Skin reactions were not found by Kawai’s method, which suggests that the citrus extract does not cause irritancy on human skin. In a wear test, a marked clinical improvement of atopic dermatitis indicated by a decrease of SCORAD index value was observed in human subjects wearing the dyed underclothes compared to the values for subjects with undyed clothes. These results imply that Citrus grandis Osbeck could be utilized as a bioactive natural material for dermal healthcare-related textiles.