Background: Varicellovirus equidalpha4 (EHV-4) is a frequent respiratory pathogen of the horse. EHV-4 sporadically induces abortion or neonatal death and although not clearly demonstrated its involvement in neurological forms is strongly suspected. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Objectives: Testing the efficacy of different compounds on EHV-4 and studying the mode of action of the most effective one. Study design: Screening and transcriptomic approach. Methods: A screening of 42 antiviral compounds was performed in vitro on E. Derm cells infected with EHV-4405/76 reference strain (VR2230). Formation of cytopathic effects was monitored by Real-Time Cell Analysis (xCELLigence and video-microscopy) and the viral load was quantified by qPCR. Results: Potential antiviral activities were confirmed for 8 molecules from the 42 compounds (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine is the most potent compound against EHV-4 in vitro with an EC50 value of 1.16 ± 0.31 μM and 0.28 ± 0.05 μM measured by xCELLigence and by qPCR, respectively. A transcriptomic analysis revealed an increase in expression of various genes involved in the interferon response. Main limitations: Study performed on one cell line with one reference strain. Conclusions: This work confirms the effect of ganciclovir against EHV-4 in vitro. The study was unable to demonstrate in vitro the antiviral activity of acyclovir against EHV-4, a molecule frequently used in the field by veterinary practitioners against equine herpesviruses. The compound with the best efficacy in inhibiting EHV-4 replication in vitro is decitabine. Transcriptomic analysis of infected cells treated with decitabine revealed activation of the innate antiviral response by stimulation of the interferon pathway. Ethical animal research: Not applicable. Informed consent: Not applicable. Competing interests: None declared. Funding: Eperon HVE4 IRCP Fund N49-2019, the IFCE (Institut Français du Cheval et de l'Equitation) grant number Rech-CS-2020-2023-023-HVE4_IRCP. CENTAURE European project co-financed by the Conseil Régional de Normandie, European Union within the framework of the ERDF-SSE operational programme 2014–2020.