The Achillea genus belongs to the Asteraceae family, which is mostly found in the northern hemisphere and is comprised of 115 species in the world. In Turkish flora, there are 52 species and 58 taxa, among them half of which are recorded as endemic. To the best of our knowledge, there has been no biological activity studied in this species until now, with the exception of one study of the antimicrobial activity of certain essential oils. This study focused primarily on the determination of antioxidant, antimicrobial, and enzyme-inhibition activity of aqueous ethanol extract of Turkish endemic Achillea cucullata by in vitro methods. The extract exhibited DPPH radical scavenging activity with an IC50 value of 132.55 ± 0.026 μg/mL, the total phenol content was 53.807 ± 0.059 (mg GAE/g), and the total flavonoid content was 21.372 ± 0.026 (mg QE/g), on the dry-weight basis. Antimicrobial activity was evaluated by a micro-dilution method focused on five microorganisms; two Gram-positive [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)], two Gram-negative [Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922)], and one fungal strain [Candida albicans (ATCC 10231)]. Results show that the MIC value for the tested microorganism was higher than 5 mg/mL. In this work, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase enzymes were strongly inhibited by the A. cucullata extract, and the IC50 values for these enzymes were 2.4 μg/mL, 0.26 μg/mL, and 24.75 μg/mL, respectively. Certain acetylcholinesterase inhibitors have been used for treatment of Alzheimer's disease in the past. α-Glucosidase inhibitors are strong drug candidates, as well as potential functional food agents, for deferring the postprandial absorbency of glucose.