ORF virus of sheep and goats is one of several zoonotic parapoxviruses. Molecular and serological diagnosis of ORF virus provides high sensitivity methods for accurate and rapid diagnosis for Orf virus infection in sheep, goat and human in Egypt. The present work aimed to isolate and characterized of Orf virus isolated from sheep, goat and human and determined the efficacy of Negilla Sativa antiviral activity. All biopsy samples from human and animals were prepared and inoculated on chorioallantoic membranes of embryonated chicken eggs for virus isolation. The isolated virus was identified and characterized by Enzyme linked immune sorbent assay, Fluorescent antibody technique, electron microscopy and polymerase chain reaction. The isolated virus give specific green fluorescence, Micrograph showed ovoid shape particles 290-300×160 nm in diameter and PCR product (B2L gene ) fragments approximately 592 bp which similar to reference Orf virus. The positive Orf virus antibodies in the serum samples by protein A ELISA, positive samples were 4 out 3, 9 out 29 and 18 out 48. Also, by IFAT were 3 out 39, 6 out 29 and 12 out 48 and by AGPT were 1 out 39, 5 out 29 and 7 out 48 in human, goat and sheep at Beni-suef Governorate, Egypt respectively. The ORF virus treated with Negilla Sativa essential oil effect on Orf virus, it reduced the virus infectivity titer from 6.9 Log10 to 1.5 Log10 by EID 50/0.2 ml. The Orf virus sensitive to the effect of temperature at 37°C and 56o C/6hr were showed reduction in the virus titer with variable Degrees. It was concluded that the PCR and protein A ELISA proved to be more rapid, simple and sensitive for detection of ORF virus infection in human and animals, Negilla Sativa essential oil has antiviral effect against Orf virus but still need extensive research for chemical composition analysis to detect active principle.