The genus Scorzonera has various medicinal values. Species belonging to this genus were traditionally used as drugs or in food. The current study aimed to determine the phytochemical composition, antioxidant activity, and biological properties of the tuber, leaf, and flower of Scorzonera undulata extracts, collected from the southwest of Tunisia. Phenolic compounds from the three parts were extracted using two solvents (water and ethanol) and two extraction techniques (maceration and ultrasound). The total phenolic content was measured by the Folin-Ciocalteu assay. Furthermore, the chemical composition of Scorzonera undulata extract was also investigated by the LC-ESI-MS method using phenolic acid and flavonoid standards. The variation of the extraction methods induced a variation in the real potentialities of the three parts in terms of bioactive molecules. However, the aerial part of S. undulata (leaves and flowers) showed, in general, the highest phenolic contents. Twenty-five volatile compounds have been detected by GC-MS in S. undulata extracts; among them, fourteen were identified before derivatization. The DPPH test showed that the aerial part of the plant has a higher antioxidant activity compared to the tuber (25.06% at 50 µg/mL for the leaf ethanolic extract obtained by ultrasound extraction). For most biological activities (anti-Xanthine, anti-inflammatory, and antidiabetic (alpha-amylase and alpha-glucosidase)), the aerial parts (flowers and leaves) of the plant showed the highest inhibition than tubers.