This study focuses on the extraction of plant species Alkanna tubulosa growing in Turkey, using solvents of different polarities (ethyl acetate, ethanol, ethanol/water, and water) and determining their chemical composition and biological activity. The extracts were subjected to qualitative LC-MS/MS analyses, which resulted in the identification of even 50 different secondary metabolites in some extracts. The existence of total phenols and flavonoids was determined. of total phenols and flavonoids was determined. The antioxidant capacity was evaluated by various in vitro assays, as well as the enzyme inhibitory capacity against cholinesterases (AChE and BChE), tyrosinase, α-amylase, and α-glucosidase, as well as human isoforms of carbonic anhydrase. In addition, the cytotoxic activity of the extracts was also examined. The results showed that the ethanol/water extract showed the strongest antioxidant activity (225.55 mg TE/g extract in ABTS and 381.14 mg TE/g extract in CUPRAC), probably due to the high total phenol (105.17 mg GAE/g extract) and flavonoids (20.12 mg RE/g extract) content, while the ethanol extract showed the strongest enzyme inhibitory potential. These chemical and biological findings provide valuable insights for potential future products based on the A. tubulosa extracts which could be used in various industries.