The alcoholic extracts of A. lutea roots from different origins (Bulgarian and Turkish) were investigated for anti-cholinesterase, anti-tyrosinase, anti-amylase, anti-glycosidase and anti-proliferative activity (against MCF-7 and MCF-10A cell lines). A validated HPLC method for the determination of two anthraquinones, two bianthraquinones, two naphthalenes, two flavonoids and one hydroxycinnamic acid was developed due to the presence of these components in the genus Asphodeline. In addition, total anthraquinones content was determined spectrophotometrically. A. lutea from Bulgaria demonstrated higher level of total anthraquinones (0.81 ± 0.05 mg g-1). A naphthalene derivative (2-acetyl-1,8-dimethoxy-3-methylnaphthalene) was the major compound in Bulgarian accession (0.96 ± 0.03 mg g-1), while caffeic acid was the main analyte in Turkish sample (0.42 ± 0.06 mg g-1). Turkish extracts were more potent as cholinesterase and tyrosinase inhibitors but less active as anti-diabetic agent. The higher anti-proliferative effect was presented by Bulgarian extract with IC50 values 120 ± 16.8 mg mL-1 (for MCF-7) and 256 ± 16.1 mg mL-1 (for MCF-10A).