Linoleic, oleic, palmitoleic, palmitic, and stearic fatty acids (FAs) are commonly used in dermatological formulations. They differ by their structure, presence in the skin, and mode of application in pharmaceuticals and cosmetics compounding. These FAs are also known as chemical penetration enhancers, but their mechanisms of penetration enhancement and effect on barrier characteristics of the skin require additional study. In this study, the authors conducted an ex vivo analysis of the distribution of lipid components in the epidermis and dermis of human skin after applying individual FAs. The goal was to elucidate possible mechanisms of penetration enhancement and FA effects on barrier characteristics of the skin. FA penetration studies were conducted ex vivo on human skin and time-of-flight secondary ion mass spectrometry (TOF-SIMS) bioimaging analysis was performed to visualize and analyze distribution of FAs in skin sections. The current study demonstrated that TOF-SIMS imaging was effective in visualizing the distribution of linoleic, oleic, palmitoleic, palmitic, and stearic acid in the human skin ex vivo after the skin penetration experiment of individual FAs. The integration of the obtained TOF-SIMS images allowed a semiquantitative comparison of the effects induced by individual FA applications on the human skin ex vivo. FAs showed varying abilities to penetrate the skin and disorder the FAs within the skin, based on their structures and physicochemical properties. Linoleic acid penetrated the skin and changed the distribution of all the analyzed FAs. Skin treatment with palmitoleic or oleic acid increased the amounts of singular FAs in the skin. Penetration of saturated FAs was low, but it increased the detected amounts of linoleic acid in both skin layers. The results indicate that application of FAs on the skin surface induce redistribution of native FAs not only in the stratum corneum layer of epidermis but also in the lipid content of full epidermis and dermis layers. The results indicate that topically applied pharmaceutical products should be evaluated for potential chemical penetration enhancement and lipid component redistribution effects during formulation.