Summary Background Skin colonization or infection with Staphylococcus aureus is known to trigger aggravation of atopic dermatitis (AD). However, the exact mechanisms by which S. aureus can worsen AD are unknown. Objective We investigated whether and how S. aureus‐derived membrane vesicles (MVs) contribute to worsening of AD. Methods Immunohistochemical and immunoelectron microscopic analyses were performed to detect staphylococcal protein A (SPA) in the epidermis of AD lesions. HaCaT cells were treated with S. aureus MVs and were analysed for the expression of cytokine genes. Immunopathology and cytokine gene profiles were analysed after topical application of S. aureus MVs to AD‐like skin lesions in a mouse model. Results The MV component SPA was detected in the keratinocytes as well as in the intercellular space of the epidermis of AD lesions colonized with S. aureus. Intact MVs from S. aureus delivered their components to keratinocytes and stimulated pro‐inflammatory cytokine gene expression in vitro. A knock‐down of Toll‐like receptor 2 or nucleotide‐binding oligomerization domain 2 using small interfering RNAs suppressed interleukin‐8 gene expression. Topical application of intact S. aureus MVs to AD‐like skin lesions in the mouse model induced massive infiltration of inflammatory cells and the resulting eczematous dermatitis. This inflammatory reaction was associated with a mixed Th1/Th2 immune response and enhanced expression of chemokine genes in AD‐like skin lesions. Conclusions and Clinical Relevance This study showed the importance of S. aureus MVs as a potent mediator for worsening of AD among many exogenous worsening factors of AD. Thus, S. aureus MVs may be regarded as one of the therapeutic targets for the management of AD aggravation.