Copper (II) oxide nanoparticles (CuO NPs) attract much attention as a promising antimicrobial agent. We studied the antibacterial properties of three types of CuO NPs against bacteria: flake-shaped particles with a diameter of 50-200 nm and a thickness of 10-20 nm (CuO-CD synthesized by chemical deposition), spherical particles with a size of 20-90 nm (CuO-EE obtained by electrical explosion), and rod-shaped particles with a length of 100-200 nm and a diameter of 30 × 70 nm (CuO-CS commercial sample). We tested how the shape, size, and concentration of the NPs, and composition of the dispersion medium affected the properties of the CuO NPs. We prepared dispersions based on distilled water, a 0.9% NaCl solution, and the LB broth by Lennox and used Triton X-100 and sodium dodecyl sulfate (SDS) as stabilizers. The concentration of NPs was 1-100 mg L. We showed that the dispersion medium composition and stabilizer type had the greatest influence on the antibacterial effects of CuO NPs. We observed the maximum antibacterial effect for all CuO NP types dispersed in water without a stabilizer, as well as in LB broth with the SDS stabilizer. The maximum inhibition of culture growth was observed under the influence of CuO-EE (by 30%) and in the LB broth with the SDS stabilizer (by 1.3-1.8 times depending on the type of particles). In the saline solution, the antibacterial effects were minimal; in some cases, the CuO NPs even promoted bacterial culture growth. SDS increased the antibacterial effects of NPs in broth and saline but decreased them in water. Finally, among the particle types, CuO-CS turned out to be the most bactericidal, which is probably due to their rod-shaped morphology and small diameter. At the same time, the concentration and aggregation effects of CuO NPs in the colloidal systems we studied did not have a linear action on their antibacterial properties. These results can be used in the development of antibacterial coatings and preparations based on CuO NPs to achieve their maximum efficiency, taking into account the expected conditions of their use.