In the present study, Zr-doped copper oxide nanoparticles (CuO-NPs) were successfully produced via the Pechini method and their structural and antibacterial properties were investigated. The synthesized powders were characterized by X-ray diffraction analysis, fourier transform infrared spectroscopy, differential scanning calorimetry with thermal gravimetry, and field-emission scanning electron microscopy equipped with energy dispersive X-ray spectroscopy. A quantitative rietveld analysis was conducted on the XRD data for structural studies. The antibacterial activity of the prepared products was evaluated by four important oral and hospital bacteria in gram-positive (Enterococcus faecalis, Streptococcus mutans) and gram-negative (Escherichia coli, Stenotrophomonas maltophilia) strains. The characterization results indicated that the Zr dopant has been incorporated into the lattice structure of the single-phase CuO powders. The resulting antibacterial evaluations showed that by increasing the Zr content in the CuO lattice, the antibacterial activity is promoted. ROS generation was determined as the predominant mechanism of the antibacterial activity of the synthesized powders. It was found that the pure and Zr-doped CuO-NPs are the promising antibacterial agents for the prophylaxis of infectious diseases, originating from oral and nosocomial bacteria. Also, the synthesis method of the prepared NPs played a key role in obtaining high antibacterial efficacy from the viewpoint of the desired size and morphology.