This research determined the uptake of individual components of topically applied microemulsions into the stratum corneum (SC) and assessed their molecular effects on skin barrier function. The microemulsions comprised oleic acid, Tween20, Transcutol and water. The effects of selected formulations, and of the individual components, on the conformational order of the SC intercellular lipids, and on SC hydration, were assessed by infrared spectroscopy. Measurements were made as a function of SC depth by progressively tape-stripping the membrane in the normal way. SC uptake of microemulsion components was quantified via extraction and analysis of the collected tape strips. SC hydration increased in proportion to the water content of the microemulsion. Each of the microemulsion components penetrated into the SC, but to different extents. Oleic acid decreased the conformational order of the SC lipids, and induced some phase separation, as revealed by the frequency shifts and peak areas of the absorbances associated with −CH2 symmetric and asymmetric stretching vibrations. Tween20 extracted some of the SC intercellular lipids. In summary, SC structure was perturbed by all components of the microemulsions, and the degree of the effects detected was proportional to the level of the respective component present in the skin.