Phenolic compounds are widely studied for their potential health benefits. However, their extraction efficiency, bioactive composition, and biological activities vary significantly depending on solvent selection and plant organ specificity. This study investigated the extraction efficiency, total phenolics and flavonoid contents, antioxidant capacity, phenolic acid and flavonoid profiles, and enzyme inhibitory activities of different extracts and fractions from the flowers and leaves of Hesperis thyrsoidea Boiss. (Brassicaceae). The extraction yield (%) varied depending on the solvents and plant organs, with crude extracts exhibiting the highest recovery, particularly in the flowers. Total phenolic and flavonoid contents were notably high in polyphenolic fractions, emphasizing the importance of selective fractionation in maximizing bioactive compound recovery. The antioxidant potential revealed that extracts of soluble free and glycoside-conjugated phenolic acid forms contributed significantly to the radical scavenging capacity. Twelve major flavonoids were detected in the samples, rutin being the most abundant in the leaves, while epicatechin and epicatechin-gallate were predominant in the flowers. The plant parts were rich in hydroxycinnamic acid derivatives, m-coumaric acid and sinapic acid exhibiting the highest concentrations in three distinct phenolic acid forms. Polyphenolic extracts exhibited particularly strong enzyme inhibitory activities for α-amylase, α-glucosidase, tyrosinase, acetylcholinesterase, and butyrylcholinesterase. These findings highlight the significance of solvent selection and fractionation techniques in optimizing phenolic compound extraction and bioactivity properties. The current results provide valuable insights concerning the development of functional foods, nutraceuticals, and pharmaceutical applications, reinforcing the potential use of H. thyrsoidea as a rich source of bioactive phenolics with strong antioxidant and enzyme inhibitory properties.