Simple SummaryAlphaherpesvirusescause genital lesions in both animals and humans. Ozone (O3) has a strong virucidal action on enveloped and naked viruses. The aim of this study was to test the in vitro virucidal and antiviral activity of an ozone/oxygen (O3/O2) gaseous mixture against caprine herpesvirus type 1 (CpHV-1). To test the virucidal activity, the virus was exposed to different concentrations (20 and 50 μg/mL) of the gaseous mixture at different time points, and a decrease in the viral titer by up to 2.0 log10 TCID50/50 μL was observed. To test the antiviral activity, the virus was exposed to different non-cytotoxic concentrations of the gaseous mixture. When MDBK cell monolayers were treated with the gas mixture after infection with CpHV-1 at a concentration of 50 μg/mL, significant antiviral activity was observed with a decrease in viral titer of 2.0 log10 TCID50/50 μL. These findings aid future studies aimed at assessing if topical treatment of genital herpes lesions in vivo with O3/O2gaseous mixture could be a valid and safe therapeutic option in an animal model, with possible translational applications in the therapy of human herpes simplex virus type 2 (HSV-2), which shares several biological similarities with CpHV-1.AbstractAlphaherpesvirusescause genital lesions and reproductive failure in both humans and animals. Their control is mainly based on prevention using hygienic prophylactic measures due to the absence of vaccines and limitations of antiviral drug therapy. Ozone is an oxidating gas showing a strong microbicidal activity on bacteria, fungi, viruses, and protozoa. The present study assessed the in vitro virucidal and antiviral activity of ozone against caprine herpesvirus type 1 (CpHV-1). The virucidal activity of a gaseous mixture containing O3at 20 and 50 μg/mL was assessed against the virus at different contact times (30 s, 60 s, 90 s, 120 s, 180 s, and 300 s). Antiviral activity of a gaseous mixture containing O3at 20 and 50 μg/mL was evaluated against the virus after 30 s and 60 s. Ozone displayed significant virucidal activity when used at all the tested concentrations whilst significant antiviral activity was observed using ozone at 50 μg/mL. The gaseous mixture, tested in the present study, showed virucidal and antiviral activity against CpHV-1 in a dose- and time contact-dependent fashion. Ozone therapy could be evaluated in vivo for the treatment of CpHV-1-induced genital lesions in goats using topical applications.