The present phytochemical investigation of the seeds of Centaurea vlachorum led to the isolation and characterisation of four compounds including two indole alkaloids N-(p-coumaroyl) serotonin (1) and moschamine (2) and two dibenzylbutyrolactone lignans matairesinol (3) and arctiin (4). This is the first report on the isolation of non-volatile secondary metabolites from C. vlachorum. The chemataxonomic significance of these compounds was summarised. Moreover, the isolated compounds were tested for their free radical scavenging activity using the following in vitro assays: (i) interaction with the free stable radical of DPPH (1,1-diphenyl-2-picrylhydrazyl), (ii) inhibition of linoleic acid peroxidation with the dihydrochloric acid of 2,2-Azobis-2-amidinopropane (AAPH). Finally, their inhibitory activity towards soybean lipoxygenase was evaluated, using linoleic acid as substrate.