In this paper, ultra-small CuO nanoparticles (NPs) were synthesized through a mechanochemical method using two different Cu-containing precursors (i.e. CuSO4·5H2O and CuCl2·2H2O), and their structure and antibacterial activity were studied. From the microstructural studies, it was observed that CuO NPs have a spherical morphology and a narrow size distribution with 7 and 14 nm median particle sizes for CuCl2·2H2O and CuSO4.5H2O precursors, respectively. The CuCl2·2H2O derived nanoparticles showed more antibacterial activity than CuSO4.5H2O derived nanoparticles. The minimum inhibitory concentration (MIC) of the synthesized nanoparticles (derived from both precursors) against E. coli and S.aureus were 3.75 and 2.50 mg/ml, respectively, which are higher than those reported in the literature for CuO NPs synthesized by other methods. This difference may be originated from ultra-small size of the synthesized nanoparticles, high bandgap energy and Fe inclusion entering from milling media and their effect on oxidative stress-mediated cytotoxicity of CuO NPs. The higher MIC value reported in this work indicates that the synthesized NPs not only show good antibacterial activity, but also they yield lower cytotoxicity, which extends their applications in the biomedical field.