The aim of the present study was to evaluate the penetration of two antifungal agents through human nail by using Photoacoustic Spectroscopy. The experiments were performed using ciclopirox in two different vehicles, resulting in formulations coded as G0291L and G0291C. The propagation rate of the two formulations through the nails was evaluated in different samples from 15 healthy volunteers. The statistical test One Way ANOVA was used to discriminate the formulation with higher penetration rate. The results showed that G0291L has the higher penetration rate. In conclusion, our results showed that Photoacoustic Spectroscopy is a useful tool to evaluate the propagation of topically applied substances through the human nail.