Fungal infection of the skin is one of the most common dermatological diseases in the world. ointment formulations are among the most suitable dosage forms for topical use to treat skin infection. Nanotechnology is a promising approach to penetrate the deeper skin layers and enhance permeability of Usnic acid (UA) through the stratum corneum. UA-loaded nanoparticles (UANP) were fabricated using the solvent evaporation method, then Nano conjugation (NC) with 2D structure of the graphene (UGNC) followed by incorporation of UGNC into ointment with water soluble base i.e. PEG400, PEG 4000, SLS, and Glycerin as the ointment forming excipient. The physical properties, in vitro drug release, and antifungal activity of UGNC nanoointment were characterized. UA NPs were spherical in shape with colloidal size <250 nm. The drug release of UGNC ointment was found to be slower than the usnic acid dispersion due to the hindrance caused by the ointment base. Nanoointment of Usnic acid NP’s Graphene nano conjugate (UGNC) show augmented effect to kill the pathogens with the triple action of combination therapy with US, NPs and their conjugate with GN. In vivo anti-fungal activity of UGNC nano-ointment on the Wistar albino rats shows significant positive outcome despite hindrance in the in vitro dissolution profile and it is better than the marketed formulation having the steroid.