Vitex trifolia L., a shrub plant, has long been used as a massage oil to treat a variety of ailments, particularly pain relief. Despite its long history of use, there has not been enough research done on its phytochemical profile and biological properties to validate its traditional use. Therefore, this current research aimed to evaluate its phytochemical profile and biological properties in two forms: Vitex trifolia L. ethanolic extract (VTE) and Vitex trifolia L. oil (VTO), which could be advantageous for clinical usage. Quality control of raw plant material, total phenolic, total flavonoid, and casticin of VTE and VTO were investigated. Furthermore, the antibacterial, antioxidant, and anti-inflammatory activities and cytotoxicity of VTE and VTO were evaluated. The study indicated that VTE contained more total phenolics (66.74±15.52 mg GAE/g), total flavonoids (29.68±2.05 mg CE/g extract), and casticin (2.45±0.02 mg/mL) than VTO. VTE consistently demonstrated stronger antibacterial, antioxidant, and anti-inflammatory properties than VTO. Taken together, VTE possessed remarkable antioxidant, anti-inflammatory, and antibacterial activities with no cytotoxic effects on cells. Even VTO had weak biological abilities. However, its stability testing revealed that the modestly accepted adjustments remained stable. Therefore, this study suggests that Vitex trifolia L. could be used as a natural product for health promotion and therapy.