In the continuation of research on Algerian medicinal plants, the leaves and flowers of Centaurea acaulis L. (an Algerian and Tunisian endemic species) were studied. The chloroform-soluble fraction of the aqueous-methanolic extract was separated and purified via chromatographic methods, yielding a new guaianolide, 14-chloro-10β-hydroxy-10(14)-dihydrozaluzanin D, along with five known sesquiterpene lactones: β-cyclocostunolide, costunolide, zaluzanin D, santamarin, and kandavanolide (compounds 1, 2, and 3 are newly reported for the genus Centaurea). Chemotaxonomic analysis revealed that C. acaulis contains santamarin (a rare eudesmanolide in the Cynareae tribe) and β-cyclocostunolide (previously unreported in Centaurea), supporting previous findings that Centaurea species typically produce germacranolides, eudesmanolides, and guaianolides often derived from zaluzanin C. Additionally, the plant was found to contain sesquiterpene lactones with significant biological activities, such as costunolide and zaluzanin D.