Ketoconazole (KTZ) is a commonly prescribed antifungal drug used to treat several tropical and systemic fungus infections. The topical bioavailability of KTZ is limited due to extremely low water solubility and high molecular weight. The present investigation aimed to develop a ketoconazole nanocrystals formulation to improve the biomimetic attributes. The ketoconazole nanocrystals were prepared using a top-down media milling technique with a size of less than 800 nm and a narrow polydispersity index (PDI) of 0.434. The simplex lattice design was used to further investigate the effect of change in the proportion of different stabilizers on critical product attributes such as particle size and PDI. The result of the in vitro drug release and anti-fungal study proved the superiority of the optimized KTZ nanocrystal in inhibiting fungal infection and suspension of pure drug. The optimized nanocrystals entrapped within Carbopol-934P gel had higher permeability through cellulose membrane compared, to the marketed product (2 % ketoconazole % w/w Ketodoc Cream®) with sustained release for 24 h. Moreover, the release profile indicated diffusion-controlled release from gel following Korsmeyer-Peppas. The present investigation successfully demonstrated the application of simplex lattice design and desirability function in optimizing ketoconazole nanocrystals to improve its transdermal application.