Vitex negundo L. is a woody aromatic shrub distributed throughout India. All parts of the plant possess a multitude of secondary metabolites and finds use for treatment of a wide spectrum of health disorders in traditional and folk medicine. The plant has anti-inflammatory, anticonvulsant, antioxidant properties and are used for the treatment of various diseases such as asthma, bronchitis, cold, cough etc. In vitro studies and phytochemical analysis will be helpful for sustainable utilization of V. negundo. This work reports in vitro propagation of the plant along with evaluation of phytochemical constituents responsible for its unique properties and antimicrobial activity. In vitro studies were carried out in MS medium with various combinations of auxin and cytokinin. Various explants were tried for inducing callus, root and multiple shoots. Phytochemical screening of secondary metabolites in V.negundo was carried out along with evaluation of antimicrobial and antioxidant potential. This study presented a detailed knowledge about phytochemical constituents, antimicrobial & antioxidant properties along with a protocol for rapid in vitro propagation of Vitex negundo.