Context: The systemic treatment of onychomycosis has been hampered by the reported side effects of antifungals in addition to the limited blood circulation to the affected nails. Topical ungual treatment would circumvent the limitations of systemic onychomycosis treatment. Objective: Preparation and characterization of nail penetration enhancer containing nanovesicles (nPEVs) loaded with sertaconazole for topical treatment of onychomycosis. Materials and methods: nPEVs were prepared using different nail penetration enhancers (N-acetyl-L-cysteine, thioglycolic acid, thiourea and ethanol) by the thin film hydration method, and characterized for their particle size, zeta potential, entrapment efficiency (EE%), elasticity, viscosity, physical stability and morphology. The selected nPEVs formula and the marketed Dermofix Õ cream were compared in terms of nail hydration, transungual drug uptake and antifungal activity against Trichophyton rubrum. Results: N-acetyl-L-cysteine was the optimum nail penetration enhancer for incorporation within vesicles. nPEVs showed high EE% of sertaconazole ranging from 77 to 95%, a size ranging from 38-538 nm and a zeta potential ranging from +48 to +72 mV. The selected nPEVs formula displayed spherical morphology and good storage stability. Compared to the conventional marketed cream, the selected nPEVs formula showed 1.4-folds higher hydration and drug uptake enhancement into nail clippings. Furthermore, it showed significantly higher zone of inhibition for Trichophyton rubrum (20.9 ± 0.25 mm) than the marketed cream (11.6 ± 0.44 mm). Conclusion: Nail penetration enhancer containing nanovesicles (nPEVs) present a very promising option, worthy of clinical experimentation on onychomycotic patients.