Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) is considered as a medicinal plant by Argentineans and Brazilians, being popularly used as sedative, eupeptic and carminative agent. The high amount of carvone produced by this species could justify its use in the treatment of some digestive disorders. The aim of this work was to evaluate the effect of the time of leaf harvesting on the composition of A. polystachya essential oil. The antimicrobial activity of this essential oil was evaluated against Escherichia coli, Staphylococcus aureus, three Candida strains and Trichophyton rubrum. Essential oils from dry and fresh leaves obtained by hydrodistillation were analyzed by gas chromatography (GC) and GC-mass spectrometry (GC-MS). The chemical profile of A. polystachya essential oils varied according to day-time harvesting of the plant material though they were basically composed of carvone and limonene. The antifungal activity of the essential oils against clinical isolates of Tricophyton rubrum, Candida albicans, C. krusei and C. famata was validated.