BACKGROUND: Origanum majorana L., belonging to the Lamiaceae family, has great potential and has been used as a folk medicine against asthma, indigestion, headache and rheumatism; in addition, the essential oils of this plant have been used widely in the food industry. Plant materials have been harvested from the Medicinal and Aromatic Plant Field of Gaziosmanpasa University. Air-dried plantmaterialswere boiled in water, filtered, and the solvent part subsequently extracted with hexane and ethyl acetate. The chromatographic method was applied to the ethyl acetate extract to isolate bioactive secondary metabolites, the structures of which were elucidated by spectroscopic techniques: basically one-dimensional and two-dimensional nuclear magnetic resonance and quadrupole time-of-flight liquid chromatography. Antiproliferative and antioxidant activities of isolated secondary metabolites were determined.