The persistent increase in the number of antibiotic resistant strains of microorganisms has led to the development of more potent but more expensive antibiotics. In most developing countries of the world these antibiotics are not readily affordable, thus making compliance difficult. This calls for research into alternative source of antimicrobials. Present work was carried out to assess the antimicrobial activity of V.negundo against some multidrug resistant pathogenic bacteria. V.negundo flower buds were collected, air dried and soxhlet extracted by using standard method for flavonoid extraction. These extracts were then tested for antimicrobial activity using disc diffusion method. MIC, MBC & TA was also calculated. flower buds bound flavonoid extract of Vitex showed highest antibacterial activity (IZ=25mm, AI=1.38±0.026) against Bacillus subtilis and (IZ=25mm, AI=1.04±0.010) against Raoultella planticola. The minimum inhibitory concentration (MIC) of Vitex was 0.039 mg/ml against Bacillus subtilis, raoultella planticola and Agrobacterium tumifaciens. The most active extract of bound flavonoid of flower buds was analyzed by GCMS study, which revealed eighty compounds in it. Two flavonoids were found in the flowers buds of Vitex negundo, as Kampferol-3-O-rutinoside (RT=30.342) and 5-hydroxy- 3,6,7,3’,4’-pentamethoxy flavones(RT=49.408), that are not has been reported earlier in flower buds of Vitex negundo. Flavonoids of flower buds extract of V.negundo Linn has the potential to be developed into an antimicrobial agent.