Weed resistances to synthetic herbicides, as well as consequent health and environmental problems, are important items to find more eco-friendly natural alternatives to weed control. Eucalyptus globulus Labill. essential oil has been traditionally used against respiratory troubles as well as insect repellent due to 1,8-cineole content. Chemical composition of commercial E. globulus essential oil and its phytotoxic activity against three common annual weeds (Portulaca oleracea L., Echinochloa crus-galli (L) Beau. and Lolium multiflorum Lam.) has been studied. Twenty-eight compounds reaching 99.83% of the total essential oil were identified by gas chromatography-mass spectrometry analysis. The oxygenated monoterpene 1,8-cineole (76.43±0.35%), followed by the monoterpene hydrocarbon α-pinene (14.64±0.27%) were the main compounds. E. globulus essential oil lacks of phytotoxicity against the seed germination of the tested weed, showing significant effect on hypocotyl and radicle elongation of E. crus-galli at the highest dose (1 µL/mL) assayed and radicle inhibitory effects at all concentrations applied (0.125, 0.25, 0.50 and 1 µL/mL) against L. multiflorum. E. globulus essential oil could be used in the management of E. crus-galli due to its post-emergence herbicidal activity.