Early biofilm formation could be inhibited by applying a thin biocompatible copper coating to reduce periprosthetic infections. In this study, we deposited crystalline Cu-doped TiO2 films using one-step DC magnetron sputtering in an oxygen atmosphere on a biased Ti6Al4V alloy without external heating. The bias voltage varied from −25 V to −100 V, and the resultant substrate temperature was measured. The deposited coatings were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), microhardness, scratch and hydrophilicity tests, potentiodynamic polarization measurements, and antibacterial assays against S. aureus and E. coli. The findings demonstrated that when a higher negative bias is applied, the substrate temperature drops, and the anatase to rutile transformation is initiated without indicating obvious Cu-containing phases. The SEM images of the films showed spherical agglomerates with homogeneously distributed Cu with decreasing Cu content as the bias value increased. Higher bias results in the grain refinement of the thinning coatings with more lattice microstrain and more defects, together with an increase in water contact angles and hardness values. Samples biased at −75 V exhibited the highest adhesive strength between coatings and substrate, whereas the specimen biased at −50 V demonstrated higher corrosion resistance. Cu-containing TiO2 coatings with pure anatase phase composition and Cu concentrations of 2.62 wt.% demonstrated excellent bactericidal activity against both S. aureus and E. coli. The layers containing 2.34 wt.% Cu exhibited very good antibacterial properties against S. aureus, only. According to these findings, the produced copper-doped TiO2 coatings have high bactericidal qualities in vitro and may be used to prepare orthopaedic and dental implants in the future.