Vitex agnus-castus is a medicinal plant of the Verbenaceae family, widely used in traditional medicine. This study is aimed at investigating the functional variability of phenolic compounds in different parts (leaves, stems, flowers, roots, and seeds) of Vitex agnus-castus methanolic extracts and at assessing their in vitro antidiabetic, antioxidant, and antibacterial activities. The results of HPLC-DAD-QTOF-MS indicated the presence of 25 phenolic compounds with a remarkable variability between plant parts; high levels were registered in chlorogenic, vanillic, 3,4-dihydroxybenzoic, and 3-hydroxybenzoic acids; hesperidin; and luteolin. V. agnus castus fruits and stems presented higher antioxidant activities. The extracts inhibited the growth of five pathogenic bacteria with MIC values documented between 7.81 and 31.25 mg/mL. In vitro antihyperglycemic effect revealed higher effect in flowers (2921.84 μg/mL) and seeds (2992.75 μg/mL) against α-glucosidase and of leaves (2156.80 μg/mL) and roots (2357.30 μg/mL) against α-amylase. The findings of this showed that V. agnus castus is a promising source for antidiabetic bioactive compounds. However, further investigations regarding the evaluation of in vivo antidiabetic effects of these compounds are needed.