Five different essential oil extractions of the aerial parts of Cleome spinosa Jacq. were examined. The oils obtained by hydrodistillation of the whole aerial parts, aerial parts without flowers (fruit, leaves and stem), flowers, fruits and leaves have been examined by GC-FID and GC-MS. The chemical profiles of the oils reveal the dominance of oxygenated sesqui- and diterpenes, with the exception of the fruit oil, which contained a high content of fatty acids. The most abundant compounds from the whole aerial parts were (Z)-phytol (31.3%), integerrimine (5.5%) and incensole (4.0%). The major compounds from the aerial portion without flowers were caryophyllene oxide (10.5%), (-)-spathulenol (7.5%) and Z-phytol (6.9%). In the flower oil, the main components were 7-alpha-hydroxy manool (23.8%), incensole (9.2%) and sclareol (8.7%). The chief constituents in the fruit oil were tetradecanoic acid (40.6%), (Z)-phytol (6.58%) and sclareol (4.5%). In the leaf oil, (Z)-phytol (19.5%), 7-alpha-hydroxy manool (6.8%) and caryophyllene oxide (4.36%) were the predominant compounds. Antimicrobial activity of the oil obtained from the whole aerial part was evaluated against nine microbial strains using a filter paper disc-diffusion method. The volatile oil showed moderate action against seven of the eight bacteria strains used, with significant inhibitory activity against Streptococcus pyogenes Group A when compared with the standard antibiotics, ampicillin and gentamicin. The fungus, Candida albicans was less sensitive to the essential oil. The oils showed moderate insecticidal activity against Cylas formicarius elegantalus, but possessed no antioxidant activity as indicated by the DPPH method. This represents the first report on the chemical composition of the essential oils from C. spinosa found in Jamaica and the in vitro antioxidant, insecticidal and antimicrobial potential of the oil from the aerial parts.