The skin permeation enhancement of many kinds of drugs and cosmetic substances by microemulsions has been widely known; however, the correlations between microemulsion microstructures and the efficiency of skin permeation are not fully elucidated. Therefore, the aim of our study was to investigate the influence of microemulsion types on in vitro skin permeation of model hydrophobic drugs and their hydrophilic salts. The microemulsion systems were composed of isopropyl palmitate (IPP), water, a 2:1 w/w mixture of Aerosol OT (AOT) and 1-butanol, and a model drug. The concentrations of surfactant mixture and model drug were maintained at 45% and 1% w/w, respectively. The concentrations of IPP and water were 15% and 39% w/w, respectively, for oil-in-water (o/w) type and vice versa for water-in-oil (w/o) type. The samples were prepared by simple mixing and characterized by visual appearance, pH, refractive index, electrical conductivity, viscosity, and determination of the state of water and IPP in the formulations using differential scanning calorimetry. Transdermal flux of lidocaine, tetracaine, dibucaine, and their respective hydrochloride salts from the drug-loaded AOT-based microemulsions through heat-separated human epidermis was investigated in vitro using modified Franz diffusion cells. The o/w microemulsions resulted in the highest fluxes of the model drugs in base form as compared with the other formulations within the same group of drugs. Moreover, the skin permeation of drug from microemulsions depended on drug molecular structure and interaction between drug and surfactant.