This report represents the first study on the chemical composition of essential oil of endemic Centaurea lycopifolia. This report also represents the first study on the anticholinesterase activity of essential oils of C. lycopifolia, C. balsamita and C. iberica. Essential oils were obtained using a Clevenger apparatus by hydrodistillation from the whole parts of C. lycopifolia, C. balsamita and C. iberica. The essential oils composition of the plants were determined by GC-FID and GC-MS (gas chromatograph/mass spectrometry) analysis. The major component of the essential oils was identified as caryophyllene oxide (9.7 %) and spathulenol (7.3 %) for C. lycopifolia, alpha-selinene (8.5 %) and hexatriacontane (8.3 %) for C. balsamita and arachidic acid (25.3 %) and hexadecanoic acid (5.9 %) for C. iberica. The essential oils of three Centaurea species indicated moderate inhibitory effect against butyrylcholinesterase and acetylcholinesterase enzyme, at 200 mu g/mL.