The chemical composition and antimicrobial activity of the essential oil from the aerial parts of Euphorbia helioscopia L. were investigated by gas chromatography-mass spectrometry (GC-MS), GC, and microdilution methods. Thirty-five compounds, representing 83.51% of the total oil, were identified. 1,6-Dihydrocarveol (31.39%), carvone (16.79%), menthol (8.23%), and trans-dihydrocarvone (5.53%) were the principal constituents of the oil. The essential oil exhibited strong antimicrobial activity against strains of the bacteria, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Shigella dysenteriae, and a strain of the fungus Candida albicans with a minimal inhibitory concentration value of 31.25 μg/mL and minimum bactericidal concentration values of 31.25 μg/mL ( S. aureus), 62.50 μg/mL ( E. faecalis), >62.50 μg/mL ( E. coli), >62.50 μg/mL ( S. dysenteriae), and >62.50 μg/mL ( C. albicans). These results indicated that the essential oil from the aerial parts of E. helioscopia could be used to control diseases caused by these microbes.