Atopic dermatitis (AD) is a complex disease characterized by chronic recurring eczema and pruritus. In addition, AD patients display increased cutaneous and systemic levels of oxidative damage markers, whose source remains elusive. In this study, we investigated oxidative and mitochondrial stress in AD epidermis. The levels of SOD2 and hydrogen peroxide are augmented in mitochondria of flaky tail (ft/ft) mouse keratinocytes, which is associated with inhibition of the glutathione system and catalase. Furthermore, reduced amounts of Gpx4 are associated with accumulation of malondialdehyde, 4-hydroxy-2-nonenal, and oxidized phosphatidylcholines in ft/ft epidermis. Cytochrome c is markedly increased in ft/ft epidermis, hence demonstrating mitochondrial stress. Topical application of MitoQ, which is a mitochondrial targeting antioxidant, to ft/ft mouse skin reduced damage to macromolecules and inflammation and restored epidermal homeostasis. Absence of alteration in the expression of SOD2, catalase, Gpx4 and limited lipid peroxidation as well as oxidized phosphatidylcholines, in the epidermis of Flg mice suggest that filaggrin deficiency marginally contributes to oxidative stress in ft/ft epidermis. Increased SOD2, lipid peroxidation and cytochrome c in the epidermis of AD patients, associated with reduced antioxidant response in primary AD keratinocytes, corroborate mitochondrial dysfunction and lack of cellular adjustment to oxidative stress in AD epidermis.