Vitex negundo(Family-Verbenaceae) is a remarkable medicinal plant with numerous therapeutic applications. In this study, phytochemical screening was performed in Vitex negundoleaf extract, and extraction optimization was performed by applying response surface methodology (RSM). Methanolic extract and microwave-assisted extraction (MAE) were found to be appropriate for obtaining the maximum yield of bioactive compounds. In single-factor analysis, solvent concentration, microwave power, and time of extraction variations were analyzed to calculate the amount of TPC, TFC, and TAC in the extract. The quantity of TPC, TFC, and TAC were fairly obtained from 60%–95% (v/v) methanol concentration, 540 -900 W microwave power, and 60–90 s extraction time. Furthermore, we also employed the response surface methodology (RSM) for optimizing microwave-assisted extraction of Vitex negundo leaves by considering three key parameters: solvent concentration, extraction temperature, and extraction duration. Condition optimization for the extraction were obtained at 95% (v/v) methanol, 720W, and 90s with TPC (227.6 mg gallic acid equivalent (GAE)/g sample), TFC (978.2 mg RE/g sample), and TAC (82.13 mg ascorbic acid equivalents/g sample). The characterization of methanol extract of Vitex negundowas done by FTIR and GC–MS to identify the various active groups and types of bioactive constituent’s presence in the extract. Subsequently, the compounds were evaluated for their bioactivity through molecular docking interactions with target proteins in silico. Furthermore, the antibacterial activity of the optimized extract was studied against two Gram +ve (Bacillus sphaericus, Pectobacterium) and two Gram-ve (Escherichia coli, Pseudomonas aeruginosa) bacteria, and the inhibitory effect was observed in all four tested bacteria. The highest antibacterial activity was shown against B. sphaericus (19± 0.087) and E. coli (19±0.064) followed by Pectobacterium (18±0.068) and Pseudomonas(13±0.093). The hemolytic activity of the optimized leaf extract (at a dose of 20µg/ml) shows a minimum hemolytic activity, i.e., 1.33± 0.06 %. It may be concluded from the study that Vitex negundo is potential and safe source of natural antioxidants that can be utilized in green therapeutics.