Fungal keratitis is a major threat to ocular morbidity and blindness. The therapeutic efficacy of eye drops against fungal keratitis is limited by their poor bioavailability, especially in patients with corneal stromal ulcers that lead to tissue defects. Therefore, intervention measures that can control fungal infection while promoting tissue regeneration are desirable. Herein, we designed and fabricated natural antifungal hydrogels that comprise a decellularized porcine cornea (DPC), gelatin and microspheres (MCs) containing voriconazole (Vor) for the management of fungal keratitis with focal corneal stromal defects. The size and structure of the Vor-loaded MCs were characterized by scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The antifungal drug Vor showed that continuous release from the hybrid hydrogel system for up to 7 days and that MCs loading did not affect the mechanical properties, in vitro release profile or cytocompatibility of the hybrid hydrogel. Moreover, the Vor-loaded hydrogels exhibited excellent antifungal properties against F. solani and A. fumigatus. The efficiency of the natural antifungal hydrogel was evaluated by an ex vivo infectious rabbit corneal defect model. After 24 h, the number of fungal colony-forming units (CFU) significantly decreased in antifungal hydrogel-treated corneal tissue compared with that in non-treated corneas and corneas treated with hydrogels without Vor. The above results demonstrated that this natural hydrogel-based drug delivery system holds great promise for preventing fungal keratitis infection while promoting focal corneal stromal regeneration. Additionally, natural hybrid hydrogels might be a candidate material for use with various drugs in the effective treatment of many other ocular diseases.