Onosma species have been used by the public for centuries to treat various diseases. This study aimed to analyze the chemical composition, antioxidant, and enzyme inhibitory activity potentials of the methanol extracts of O. cappadocica and O. rutilum, endemic to Turkish flora. The chemical compositions of the extracts were determined by using spectrophotometric and chromatographic methods. The biological activities of the extracts were determined by using antioxidant and enzyme inhibitory test systems. It was determined that the extracts contain high amounts of hesperidin, hyperoside, and rosmarinic acid. While O. rutilum exhibited higher activity in phosphomolybdenum, ABTS radical scavenging and ferrous ion chelating assays, reducing power, and DPPH radical scavenging tests resulted in the superiority of O. cappadocica. O. rutilum exhibited higher inhibition activity on all enzymes (acetylcholinesterase, butyrylcholinesterase, tyrosinase, and alpha-amylase), except for alpha-glucosidase. It was concluded that the extracts in question could be used as alternative agents in the food, cosmetic and medical industries due to their antioxidant and enzyme inhibitory activities.