Lithraea molleoides (Vell.) (Anacardiaceae) is a tree found in Brazil, Paraguay, Bolivia, Uruguay, Argentina and Chile. It is popularly used in the form of alcoholic extracts, decoctions and infusions for the treatment of cough, bronchitis, arthritis, diseases of the digestive system and as diuretic, tranquilizer, haemostatic and tonic agent. The objectives of this study were the extraction of the essential oil of the mature fruit, leaves and aerial parts of the plant and quantification of the yield thereof; the identification and quantification of the principal components of the essential oil and the determination of its antimicrobial activity against bacteria and yeast. The essential oil yield of the mature fruits was 1.0% but essential oil was not detected in leaves and flowery branches. The gas chromatograph-mass spectrometer (GC-MS) analysis of the essential oil of the mature fruits detected the presence of limonene (89.89%), α-pinene (3.48%), β-pinene (2.63%), α-terpineol (1.27%), myrcene (0.64%), sabinene hydrate (0.54%), 4-terpineol (0.28%), camphene (0.22%) and Δ-3-carene (0.13%). The essential oil was active against just a few of the Gram-positive bacteria and yeast tested and did not present antimicrobial activity against Gram-negative bacteria.