Background/Objectives: Vitex agnus-castus L. is a well-known medicinal herb shown to be effective in treating gynecological disorders. However, no systematic comparative studies have been conducted between V. agnus-castus leaf extract (VACLE) and Vitex agnus-castus seed extract (VACSE). We performed gas chromatography–mass spectrometry (GC–MS) analysis of VACLE and VACSE and measured total phenolic and flavonoid contents. Methods: The bioactivity testing included antioxidants, antibacterial, antifungal, anticancer, and antidiabetic activities. Results: A total of 55 GC–MS compounds were identified in VACLE and 34 in VACSE; isoamyl formate (27.96%) and a pyranone derivative (14.11%) were detected exclusively in VACLE, whereas cis-linoleic acid (40.58%) and palmitic acid (21.87%) predominated in VACSE. VACLE showed significantly higher TPC (94.12 vs. 54.12 mg GAE/g DW) and TFC (82.00 vs. 42.00 mg QE/g DW). The VACLE demonstrated moderate antioxidant activity and generally stronger bioactivity than VACSE, as evidenced by its lower ABTS⁺ radical scavenging IC₅₀ value (55 vs. 70 μg/mL), antibacterial activity (MIC: 6.25–50 vs. 12.5–100 μg/mL), anticancer activity against HepG2 cells (IC₅₀: 93.2 vs. 247.5 μg/mL), and antidiabetic activity through α-amylase inhibition (IC₅₀: 28.7 vs. 70.1 μg/mL). VACSE exhibited greater antifungal activity than VACLE against all tested Candida strains, with the highest activity observed against C. parapsilosis (MIC: 6.25 ± 2.26 μg/mL). VACLE induced transcriptional changes consistent with caspase-mediated apoptosis, characterized by increased expression of caspase-8, caspase-9, and Bax and decreased expression of Bcl-2/Bcl-xL, pending protein-level confirmation. Conclusions: In conclusion, VACLE exhibits notable antioxidant, antibacterial, anticancer, and antidiabetic properties, whereas VACSE shows greater antifungal activity. These findings highlight tissue-specific differences in phytochemical composition and in vitro biological activities and provide a basis for future studies involving compound isolation, mechanistic validation, toxicity assessment, and in vivo evaluation.