PURPOSE: Herpes keratitis (HK) is the leading cause of cornea-derived and infection-associated blindness in the developed world. Despite the availability of effective antivirals, some patients develop refractory disease, drug-resistant infection, and topical toxicity. A nonpharmaceutical treatment modality may offer a unique advantage in the management of such cases. This study investigated the antiviral effect of nonthermal dielectric barrier discharge (DBD) plasma, a partially ionized gas that can be applied to organic substances to produce various biological effects. METHODS: Human corneal epithelial cells and explanted corneas were infected with herpes simplex virus type 1 (HSV-1) and exposed to culture medium treated with nonthermal DBD plasma. The extent of infection was measured by plaque assay, quantitative PCR, and Western blot. Corneal toxicity assessment was performed with fluorescein staining, histologic examination, and 8-OHdG detection. RESULTS: Application of DBD plasma-treated medium to human corneal epithelial cells and explanted corneas produced a dose-dependent reduction of the cytopathic effect, viral genome replication, and the overall production of infectious viral progeny. Toxicity studies showed lack of detrimental effects in explanted human corneas. CONCLUSIONS: Nonthermal DBD plasma substantially suppresses corneal HSV-1 infection in vitro and ex vivo without causing pronounced toxicity. TRANSLATIONAL RELEVANCE: Nonthermal plasma is a versatile tool that holds great biomedical potential for ophthalmology, where it is being investigated for wound healing and sterilization and is already in use for ocular microsurgery. The anti-HSV-1 activity of DBD plasma demonstrated here could be directly translated to the clinic for use against drug-resistant herpes keratitis.