Background and Objectives: As one of the major skin fatty acids, cis-6-hexadecenoic acid (C16:1Δ6) exhibits a specific antibacterial activity and might play a specific role in the defense mechanism against Staphylococcus aureus, in healthy subjects whereas S. aureus frequently colonizes the skin of patients with atopic dermatitis (AD). Methods: Fatty acid composition of sebum at the recovery level was analyzed by gas chromatography and S. aureus colonizing the skin was assessed by the ‘cup-scrub’ method (9 patients and 10 healthy controls). To evaluate in vivo effect of C16:1Δ6 on colonization, C16:1Δ6 was applied for 2 weeks on the upper arm skin of another group of AD patients (11 patients). Results: Analysis of sebum lipids revealed that there is a significant lower free C16:1Δ6 content in nonlesional skin from AD patients compared with healthy controls. This lower content is also accompanied by a significantly lower level of C16:1Δ6 in the total fatty acid composition of sebum (analyzed following hydrolysis). The lower level of free C16:1Δ6 correlated significantly (R2 = 0.41, p < 0.01) with the numbers of S. aureus colonizing nonlesional skin. Topical application of free C16:1Δ6 on the skin of AD patients for 2 weeks abolished the markedly increased bacterial count in 6 out of the 8 AD patients tested. Conclusions: Free C16:1Δ6 may be involved in the defense mechanism against S. aureus in healthy skin and this deficit triggers the susceptibility of the skin to colonization by S. aureus in AD.