One of the most common fungal skin infections is candidosis. Topical application of drugs at the pathological sites offers potential advantage of direct drug delivery to the site of action. The main aim of this work was to evaluate an optimal nystatin nanoemulsion for topical application avoiding undesirable side effects as systemic absorption and toxicity. Surface morphology and droplet size distribution of nystatin nanoemulsion was determined by transmission electronic microscopy and dynamic light scattering. Vertical diffusion Franz-type cells and high-performance liquid chromatography were used to perform the in vitro release and ex vivo human skin permeation studies. Transdermal permeation parameters were estimated from the permeation values using different theoretical approaches. Microbiological studies were performed to evaluate the antifungal effect. Nanoemulsion exhibited a spherical shape with smooth surface and mean droplet size between 70 and 80 nm. The pharmacokinetic release showed the nanoemulsion is faster than commercial ointment Mycostatin® improving the potential therapeutic index. Permeation studies demonstrated nystatin was not absorbed into systemic circulation and the retained amount in the skin was sufficient to ensure an antifungal effect. This antifungal effect was higher for nystatin loaded nanoemulsion than nystatin itself. A therapeutic improvement of the nystatin nanoemulsion treatment compared with the classical ones was achieved.