Background Atopic dermatitis (AD) is a composite disease characterized by derangement of the skin permeability barrier (SPB), altered immune defence, and dysbiosis. Little is known on the role played by the sebaceous gland (SG) activity in the SPB integrity and in the AD pathomechanisms. Objectives To investigate profiles of sebaceous and epidermal free fatty acids (FFAs), squalene, cholesterol, triglycerides (TGs), and wax esters (WEs) in sebum and stratum corneum (SC) from seborrheic and non-seborrheic areas, in healthy subjects and patients with AD. To simultaneously acquire aminoacidome in SC. Methods In healthy controls and patients with AD, sebum and SC were sampled consecutively from facial areas (forehead, and cheeks). SC was sampled also from non sebaceous areas (arm) in healthy controls and from the non lesional and lesional areas on the arm in AD. Sampling was preceded by assessments of skin biophysics, i.e. TEWL and corneometry. Results Disruption of the SBP was associated with decreased levels of lipids of both sebaceous and epidermal type. Extent of lipid derangement in the SG and the SC was correlated with the AD severity. Relative composition of natural moisturizing factors was altered in the SC of patients with AD. Conclusions The SG activity is compromised in adult AD. Aminoacidome is deranged in the facial areas in AD. Lipid signatures in association with aminoacidome, and skin physical properties may serve the definition of phenotype clusters that associate with AD severity.