Fungal keratitis is a major contributor to monocular blindness, globally. This is because, application of conventional topical therapy yields poor outcomes owing to the poor penetration and fungistatic nature of these antifungal agents. Cell penetrating peptides (CPPs) with potent antifungal effect may provide a solution to address these challenges. In this study, the antifungal efficacy of two CPPs: a corneal-targeting peptide (CorTS 1) and a non-targeting peptide (Tat2) was evaluated. Both peptides effectively inhibited the growth of Fusarium dimerum hyphae by modulating membrane permeability in vitro. Additionally, these peptides demonstrated successful trans-epithelial penetration in rabbit eyes and exhibited superior therapeutic effects compared to commercially available natamycin ophthalmic suspension in mouse model of Fusarium keratitis. While Tat2 showed greater antifungal potency, its non-specific targeting and anti-inflammatory properties suggest its potential utility in early-stage fungal keratitis. In contrast, CorTS 1, with its corneal-targeting capability, may be more effective for treating late-stage deep stromal keratitis. These findings highlight the potential of biologically active CPPs as promising new therapies for fungal keratitis.