Achillea biebersteinii and Anthemis tinctoria, which are widely distributed species of the Asteraceae family, are used in folk medicine in the form of herbal tea or extract in the treatment of many diseases. The aim of this study was to investigate the chemical content, antimicrobial, antioxidant, enzyme inhibitor activities and cytotoxic effects of 80% ethanol extract of these two species and make a comparative analysis. In accordance with the data obtained, the major component of A. biebersteinii was determined as Cyclododecane (14.47%), while that of A. tinctoria was determined as Phytol (23.15%). A. biebersteinii, which showed moderate activity in terms of antimicrobial activity, produced more active inhibition than A. tinctoria did. Both plants showed high levels of antioxidant activity. The total phenol and total flavonoid contents of A. tinctoria were higher than those of A. biebersteinii. It was determined that there was no significant activity when the extracts were compared with galanthamine, which is the reference drug in terms of enzyme inhibitory activity. When the in vitro anticancer activity of human breast cancer cell line was examined, it was determined that A. tinctoria had a cytotoxic effect at high concentrations (IC50;0.82mg/mL), and A. biebersteinii showed strong cytotoxicity at all concentrations (IC50;<0.0625mg/mL). These two plants of the same family were evaluated in terms of many different biological parameters and it was revealed that A. biebersteinii was more active than A. tinctoria. However, in vivo studies are needed to determine whether these plants can be used as phytotherapeutic agents.