In present study, copper oxide nanoparticles (CuONPs) synthesized by quick and eco-friendly phytogenicreduction of copper salt (copper sulphate CuSO4.H2O) solution with Pterocarpus marsupium extract. UV-VIS spectrometry indicated formation of nanoparticles via absorption spectra of copper colloidal solution at 442 nm. Phytosynthesis of CuONPs were further characterized by Transmission electron microscopy; scanning electron microscopy and Fourier transform infrared spectroscopy. The experimental results showed that diameter of CuONPs in colloidal solution were < 40 nm. Further, antibacterial activities of CuONPs were determined against Gram negative Escherichia coli- MTCC-9721, Proteus vulgaris- MTCC-7299, Klebsiella pneumonia- MTCC-9751 and Gram positive i.e. Staphylococcus aureus- MTCC-9442, Staphylococcus. epidermidis- MTCC- 2639, Bacillus cereus- MTCC-9017 bacteria by well agar diffusion and microdilution method. Notably, The CuONPs showed an effective antibacterial activity against all test microorganisms where K. pneumonia and E.coli showed maximum ZOI and MIC respectively i.e. 24 mm and 6 μg/ml.