Volatile constituents of the essential oil and n-hexane extract of Jacaranda acutifolia Humb. and Bonpl. (Bignoniaceae) leaves were determined, and their antimicrobial activities were investigated using an agar diffusion method. The minimum inhibitory concentrations (MIC) were determined and compared with those of standard antibiotics (penicillin, gentamicin and nystatin). The chemical composition of the oils was analyzed by capillary gas chromatography (GLC-FID) and gas chromatography–mass spectrometry (GLC-MS). Thirty-four components, comprising almost 93.8% of the total peak area, were identified in the leaf essential oil. The main components were methyl linolenate (26.7%), 1-octen-3-ol (10.8%), methyl phenyl acetate (9.9%), β-linalool (5.5%) and palmitic acid (4.7%). The n-hexane extract revealed similar oil constituents, but also p-benzoquinone, phenyl acetic acid, resorcinol and homogentisic acid. The oil showed some activity against Staphylococcus aureus and Escherichia coli with MIC values of 2.2 and 2.9 mg/mL, respectively, and moderate activity against Candida albicans, Salmonella typhimurium and Shigella flexneri. The n-hexane extract showed moderate activities against all tested microorganisms, with MIC values ranging from 3.5 to 10.2 mg/mL. The antimicrobial activities of the hydrodistilled and extracted leaves make their local traditional uses rational.