The Lamiaceae family has approximately 300 genera and 7,500 species and it is estimated that at least 22 genera and 402 species. Leptohyptis macrostachys(Benth.) Harley & J.F.B. Pastore is a shrub species belonging to the family Lamiaceae, found in the semi-arid region of northeastern Brazil and popularly known as "alfavaca-brava" and "hortelã-do-mato". In this study, a chemical characterization of L. macrostachysessential oil was carried out. The plant material was collected in the city of Serra Branca, Paraíba, in northeastern Brazil. The essential oil from L. macrostachyswas obtained by the hydrodistillation process. The chemical characterization of the essential oil was performed by a gas chromatograph coupled to a mass spectrometer (GC-MS). An analysis of the antimicrobial activity of the essential oil was carried out using the broth microdilution method to determine the Minimum Inhibitory Concentration (MIC), using Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, S. epidermidis, Candida albicans, C. tropicalisand Cryptococcus neoformansstrains. The investigation of the essential oil from L. macrostachysby GC-MS allowed the identification of 43 components, corresponding to 99% of the total oil. The major constituents were Fenchone (20.14%), 1,8-cineole (19.74%), β-pinene (12.31%), Germacrene D (7.14%), Limonene (6,74 %), α-pinene (4.74%) and Thymol (4.52%). In relation to the antimicrobial activity, the essential oil from L. macrostachyspresented a strong activity, being able to inhibit bacterial and yeast growth in concentrations below 600 μg/mL. Among the bacterial strains, the essential oil showed a greater activity against the gram-positive strains and among the microorganisms used in this study, the essential oil presented higher potency against yeasts than against bacteria strains.