Objectives: Tanacetum L. belongs to Asteraceae family and is represented by 46 species in Türkiye. Tanacetum genus is known for its insecticide and insect repellent effect. T. argyrophyllum contains sesquiterpene lactone derivatives. These compounds are responsible for its various activities, especially cytotoxic, antitumor, phytotoxic, antimicrobial, antiviral, and antifungal activity. There are not enough biological activity studies on the plant that are likely to have a wide variety of activities in terms of the compounds it contains. The aim of the present study is to evaluate various biological activities of 80% aqueous methanol extract prepared from aerial parts of T. argyrophyllum (K. Koch) Tzvelev var. argyrophyllum collected from Sivas province of Turkey. Materials and Methods: Antioxidant activity of the methanol extract was determined by 2,2’-diphenyl-1-picryl-hydrazyl and 2,2-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) radical scavenging activity, total phenolic, and total flavonoid content tests. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were investigated via Ellman’s spectrometric method. Results: Total phenolic content was found as 71.67 mg/gallic acid equivalent g and total flavonoid content was 25.225 mg/quercetin equivalent g on a dry extract weight basis. In this work, AChE, BChE, and α-glycosidase enzymes were inhibited by the extract of T. argyrophyllum var. argyrophyllum. IC50 values for these enzymes were found as 266.79 µg/mL for AChE and 176.91 µg/mL for BChE. Also, the α-glycosidase activity exhibited a dose dependent manner with increasing concentration. Conclusion: According to the results, T. argyrophyllum var. argyrophyllum can be used as an ingredient of functional foods as well as herbal products for diabetic and Alzheimer’s disease patients.