This study was conducted to characterise phytochemicals and to explore the biological activities of Vitex negundo leaves. The washed, course powder of V. negundo leaves were extracted with different solvents of increasing polarity. All the extracts were characterized and biological activities were compared. The results revealed that the ethanolic and cold water extracts showed the presence of all phytochemicals studied except protein compared to other extracts. Further, the quantitative estimation of phytochemicals showed that the ethanolic extract had highest yield and maximum amount of total polyphenols, flavonoids, and alkaloids with the least amount of tannins compared to other extracts studied. Furthermore, the highest total polyphenol content corresponds with the potent biological activities. Indeed, in vitro antioxidant and antisteroidogenic activities were highest in the ethanolic extract than others. To conclude, the present study is the first to report the characterization and antiandrogenic property of V. negundo leaf extracts. The ethanolic extract of V. negundo leaves can be used as an antioxidant and antiandrogenic agent. Hence, it can be considered for the treatment of hyperandrogenic conditions like polycystic ovary syndrome, etc.