CuO nanoparticles (NPs) were added to cement matrices in quantities of 0.25, 0.50 and 1.00 wt% to inhibit the growth of Gram-positive (Bacillus cereus,Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa,Escherichia coli) bacteria. It was shown that CuO NPs, in all tested concentrations, improved the antibacterial properties of the cement matrix. Nevertheless, the best mechanical, structural and durability properties were obtained for cement composites doped with CuO NPs at 0.25 wt%. Larger amounts of NPs caused a decrease in all parameters relative to the reference mortar, which may be the result of a slight change in the porosity of the composite microstructure. For 0.50 wt% CuO NPs, a slight increase in the volume of micropores in the cement matrix was observed, and an increased number of larger pores was confirmed by non-invasive computed tomography (CT). The reduction in the mechanical parameters of composites with 0.50 and 1.00 wt% CuO NPs may also be due to the slower hydration of the cement binder, as confirmed by changes in the heat of hydration for these configurations, or agglomeration of NPs, especially for the 1.00 wt% concentration, which was manifested in a decrease in the plasticity of the mortars.