In this study, the essential oil of Citrus aurantifolia (Christm.) Swingle was extracted by hydrodistillation and, after chemical characterization, was evaluated for its antimicrobial activity on a representative range of ATCC and clinical strains. The chemical composition of the volatile fraction of key lime oil was determined using gas chromatographyâmass spectrometry (GCâMS) and GCâflame ionization detection (GCâFID). The antimicrobial activity of the oil was evaluated on a representative range of Gram-positive bacteria, Gram-negative bacteria and yeasts, including ATCC and clinical strains, by the disc diffusion method and the broth microdilution method. The major components of lime essential oil proved to be Î²-pinene (12.6%), limonene (53.8%), Î³-terpinene (16.5%), terpinolene (0.6%), Î±-terpineol (0.4%) and citral (2.5%), which are very likely responsible for the good antimicrobial activity, in particular on Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis and Staphylococcus epidermidis).