Five nodular samples were collected from clinically suspected cattle for lumpy skin disease (LSD) from five governorates in Egypt during 2020. Real-time PCR confirmed the presence of lumpy skin disease (LSDV) in all samples. One sample was isolated and passaged three times on the chorio-allantoic membrane (CAM) of specific pathogen free embryonated chicken eggs (SPF-ECEs), resulting in the formation of pock lesions. The isolated sample underwent partial gene sequencing of the G-protein coupled chemokine receptor (GPCR) gene for molecular characterization, revealing its close relation to circulating LSDV strains in Africa and the Middle East. Subsequently, zinc oxide nanoparticles (ZnONPs) were synthesised using a green method with zinc sulphate heptahydrate as a precursor and Arabic gum as a stabilizer. Characterization of ZnONPs using Fourier transform spectroscopy (FT-IR), zetasizer, X-ray diffraction (XRD), and transmission electron microscopy (TEM) showed a size range of 23.6 to 38.0 nm and a surface charge of -25.7 mV. The cytotoxicity of ZnONPs was evaluated on human oral epithelial cells (OEC), showing that the survival rate of cells decreased from 100.99 to 90.23% at concentrations of 0.31 and 50 mg/ml respectively. ZnONPs were used to inhibit LSDV replication in Madin Darby bovine kidney (MDBK) tissue culture using plaque reduction assay showing 50% inhibitory concentration (IC50) at 45.36 µg/ml and cytotoxicity (CC50) at 30.9 µg/ml with a selectivity index of 0.68. Furthermore, TEM imaging confirmed the antiviral efficacy of ZnONPs against LSDV. These findings demonstrate the antiviral effect of ZnONPs against LSDV, offering promising applications in veterinary medicine field.