The interest in many traditional natural products is increasing. Natural products continue producing bioactive agents owing to the remarkable available chemical diversity. They were evaluated as prospective therapeutic candidates for the treatment of human and animal infectious diseases. Euphorbiaceae, the spurge family, holds a significant place in the domain of plant families, with scientific evidence of antiviral, antibacterial, anticancer, cytotoxic and antitumor properties. In this regard, the current study intends to investigate the antibacterial, antifungal, antiviral and cytotoxic properties of Euphorbia greenwayi var. greenwayi Bally & S. Carter. The dried aerial parts of E. greenwayi var. greenwayi Bally & S. Carter were used, then extracted with 70% ethanol, solvent was distilled off till dryness. The antimicrobial activity of the extract and both MIC and MBC were evaluated against one strain of Gram-positive bacteria: Staphylococcus aureus ATCC9144; four strains of Gram-negative bacteria: Klebsiella pneumonia ATCC10031, Escherichia coli ATCC10536, Salmonella typhi ATCC14028, Pseudomonas aeruginosa ATCC9027and yeast: Candida albicans ATCC10231. The antiviral activity of hydroalcoholic extract against Rotavirus infection was determined as well as the cytotoxic properties. The antibacterial examination revealed potential activity of the hydroalcoholic extract against all tested species with the inhibition zone ranged from 14.7 to 29.7 mm. The highest activity was against S. aureus and C. albicans. MIC and MBC results proved that the extract is potentially bacteriostatic and bactericidal agents against both Gram-positive and Gram-negative bacteria and against the tested yeast. Also, the extract has the ability to prevent Rotavirus attachment with the cell host. This research revealed that the hydroalcoholic extract of aerial parts of E. greenwayi var. greenwayi Bally & S. Carter has significant antimicrobial potential that can be implemented in different pharmaceutical formulations.