Drug delivery into hair follicles with the use of nanoparticles (NPs) is gaining more importance as drug-loaded NPs may accumulate in hair follicle openings. The aim was to develop and evaluate a pluronic lecithin organogel (PLO) with roxithromycin (ROX)-loaded NPs for follicular targeting. Polymeric NPs were evaluated in terms of particle shape, size, zeta potential, suspension stability, encapsulation efficiency and in vitro drug release. Lyophilized NPs were incorporated into the PLO and rheological measurements of the nanoparticles-embedded organogels were done. The fate of the NPs in the skin was traced by incorporation of a fluorescent dye into the NPs. As a result, ROX was efficiently incorporated into polymeric NPs characterized by the appropriate size (approximately 300 nm) allowing drug delivery to hair follicles. In ex vivo human skin penetration studies, horizontal skin sections revealed fluorescence deep in the hair follicles. Although the organogel has higher affinity to the lipidic follicular area than an aqueous suspension of NPs, it did not seem to improve penetration of the NPs along the hair shaft. The results proved that it was possible to achieve preferential targeting to the pilosebaceous unit using polymeric NPs formulated either into the aqueous suspension or semisolid topical formulation.