Inorganic metal oxide nanoparticles of zinc, copper and iron have been widely studied for their use in biomedical research due to their novel physical, chemical and biological properties. Present work involves studies on copper oxide nanoparticles for their antibacterial activity. Copper oxide (CuO) nanoparticles were synthesized by template-free hydrothermal synthesis using different precursors of copper (nitrate, acetate, chloride and sulphate). The samples were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis, ultraviolet-visible (UV-Vis) absorption spectroscopy, scanning electron microscopy (SEM) and zeta potential analysis. The XRD patterns of the synthesized samples show presence of (−111) and (111) planes, which can be assigned to the monoclinic phase of CuO with the average crystallite size ranging between 43 and 49 nm. The SEM reveals rod-shaped morphology of the samples with a broad size distribution. The optical band gap calculated by UV-Vis absorption study ranges between 1.35 and 1.38 eV. The highly negative zeta potential values of the CuO synthesized with nitrate (−23.78 ± 0.95 mV), acetate (−11.86 ± 1.06 mV) and sulphate (−22.33 ± 0.85 mV) precursors of copper reveal the good colloidal stability of the synthesized particles. While CuO synthesized with copper (II) chloride shows intermediate stability with the zeta potential as 9.40 ± 1.86 mV. The antibacterial activity of the synthesized CuO was studied against human pathogens like Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacterial strains, which are proved to be efficient and precursor-dependent. The minimum inhibitory concentration of CuO against E. coli and S. aureus were found to be 0.15 and 0.1 mg ml −1 , respectively. CuO nanoparticles exhibit good antibacterial activity, hence, it has potential for biomedical applications in preventing and treating infections.