Multidrug resistance (MDR) is an emerging global health concern worldwide, driving the need for innovative solutions. Herbal approaches are gaining attention and acceptance due to safer profiles and very few side effects. In this study, silver nanoparticles (VN-AgNPs) were synthesized using Vitex negundo, a medicinally valuable plant. A methanolic extract was prepared from Vitex negundo and the phytochemical evaluation confirmed the presence of flavonoids, alkaloids, and terpenoids, with quantitative analysis revealing high total phenolic content (TPC: 23.59 mg GAE/g) and total flavonoid content (TFC: 45.23 mg rutin/g), both maximized under microwave-assisted extraction (MAE). The antioxidant activity was also highest (18.77 mg AA/g). Characterization of the optimized extract by GC–MS identified various bioactive compounds. VN-AgNPs were synthesized using the aqueous leaf extract under specified conditions and were structurally characterized using many techniques and evaluated for antibacterial activity against four bacterial strains. VN-AgNPs exhibited significant antibacterial efficacy with inhibition zones measuring 16 ± 0.87 mm against Bacillus (Gram-positive), 15 ± 0.46 mm against E. coli (Gram-negative), 12 ± 0.64 mm against Pseudomonas (Gram-negative), and 11 ± 0.50 mm against Pectobacterium (Gram-negative plant pathogen). These findings highlight the efficacy of green-synthesized VN-AgNPs as a promising alternative to combat MDR pathogens, paving the way for sustainable and effective antimicrobial strategies.