Micro- and nanoemulsions are potential drug solubilizers and penetration enhancers through the high surfactant/co-surfactant content. This study aimed to evaluate the influence of minoxidil (MXD) solubilized in the microemulsions (MEs) on drug release by in vitro/ex vivo diffusion through the semi-permeable membrane Spectra/Por®
(Spectrum Laboratory, Gardena, CA, USA) and porcine ear skin. Moreover, a residual amount of drug in the skin after ex vivo diffusion was evaluated. The reference MER
, lecithin-containing MEL
, and gelatin-containing MEG
were characterized in terms of their size, polydispersity index, density, viscosity, electrical conductivity and surface tension. Based on the in vitro diffusion, it can be argued that MEL
slowed down the drug release, while MER
have no significant effect compared to the sample, in which propylene glycol (PG) was used as a solubilizer. Determination of the residual drug amount in the skin after 6 h of the ex vivo permeation was demonstrated as the most valuable method to evaluate the effectiveness of the ME’s application. The results indicate that the most optimal MXD permeation enhancers in alginate gel were the natural surfactants containing MEs. MXD solubilization in MEG
had caused more than 5% of the drug remaining in the skin, which is almost a 1.5-fold higher amount compared to the reference gel.