Fungal hyphae deeply invade the cornea in fungal keratitis. The corneal stroma hinders the infiltration of antifungal drugs and reduces their bioavailability. Here, this work reports a peptide conjugate nano‐assembly that permeates the stroma and kills the pathogen without irritating the ocular cornea. The hydrophilic surface of the nano‐assembly ensures deep permeation into the stroma. When encountering a fungal hyphal cell, the nano‐assembly disassembles and exposes the α‐helical peptide to destroy the fungal membrane, thus inactivating the pathogen. In a rabbit model of fungal keratitis, the nano‐assembly exhibits a better therapeutic effect than commercially available natamycin ophthalmic suspension. Peptide conjugates with a nano‐assembled structure and assembly–disassembly behavior could serve as the foundation of a new therapy for fungal keratitis.