Evaluated the antibacterial effect of aqueous and ethanolic extract of the leaf, bark of Vitex negundo L. against previously characterized Staphylococcus aureus and Streptococcus pyogenes was analyzed in this study. Aqueous and ethanolic extract of the leaf, bark of Vitex negundo L. were prepared with help of soxhlet unit. Further, evaluated the antimicrobial activity of these extract were analyzed against S. aureus and S. pyogenes. Aqueous and ethanolic extract of the leaf and bark of V. negundo showed significant antibacterial activity against Staphylococcus aureus and Streptococcus pyogens. 35 μg/ml aqueous extract of leaf of the V. negundo showed 207.02, 108.20% antimicrobial activity against Staphylococcus aureus and Streptococcus pyogens respectively as compared to the control. Similarly, ethanolic extract of leaf of V. negundo showed significant values against S. aureus and S. pyogens. 35 μg/ml ethanolic extracts of leaf of V. negundo showed 120.16% more inhibition zones against S. aureus. The aqueous and ethanolic extract of bark of the V. negundo also showed antimicrobial activity against S. aureus and Streptococcus pyogens. 35 μg/ml aqueous extract of bark of V. negundo showed 96.77% inhibition zone against S. aureus as compared to control. Ethanolic extract of bark of V. negundo showed significant against Staphylococcus aureus and Streptococcus pyogens. 15, 20, 25, 30, 35μg/ml extract showed more inhibition zone by 18.81, 59.40, 138.61, 217.82, 257.42 % respectively against Staphylococcus aureus as compared to 15μg/ml norflox (control drug). Aqueous extract of the leaves as well as barks of V. negundo exhibited 10 μg/ml MIC against S. aureus and S. pyogenes.