This work presents results on the influence of thickness on the structure and biological response of Cu-O coatings deposited on commercially pure titanium (cpTi) substrates using direct current (DC) magnetron sputtering. The deposition times were 5, 10, and 15 min to obtain coatings with different thicknesses. The results show that the films deposited for 5, 10, and 15 min correspond to thicknesses of 41, 74, and 125 nm, respectively. The phase composition of the coatings is in the form of a double-phase structure of CuO and Cu2O in all considered cases. The roughness is on the nanometric scale and no obvious trend as a function of the thickness can be observed for the deposited films. Also, it was found that, with an increase in the thickness of the films, the distribution of the heights becomes closer to symmetrical. The antimicrobial efficacy of different Cu-O-coated cpTi substrates was examined using a direct contact experiment. A possible bactericidal effect was investigated by inoculating a 200 μL bacterial suspension on CuO-coated cpTi and cpTi (control) for 24 h at 37 °C. The results showed that Cu-O-coated cpTi substrates have a 50%–60% higher antimicrobial activity than the substrate. At the same time, human osteosarcoma (MG-63) cells growing on Cu-O-coated cpTi substrates showed 80% viability following 24 h incubation. Depending on magnetron sputtering process parameters, a different coating thickness, various crystallite phase compositions, and diverse biocompatibility were obtained.