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Surface modification of the Ti6Al4V alloy (ASTM grade 5), with the fabrication of vertically oriented TiO₂ nanotubes, has been receiving increasing attention both as a way to provide advanced bioactive features and the ability to act as reservoirs for a localized, controlled drug release. In this work, TiO₂ nanotubes were grown on the surface of a Ti6Al4V alloy through electrochemical anodization. An ethylene glycol-based electrolyte containing 0.5 wt.% NH₄F and 2.5% (<i>v</i>/<i>v</i>) H₂O was used. Post-anodizing heat treatments at 500 °C in air atmosphere were performed to achieve a crystalline oxide layer with a higher mechanical stability. Following these treatments, Zn or Cu nanoparticles were incorporated into the nanotubular structures through electrodeposition processes. Then, the antimicrobial performance of the obtained surfaces was assessed against <i>Staphylococcus epidermidis</i>, a Gram-positive bacterium common in implant-related infections. Lastly, the cytotoxicity of the produced surface was evaluated against MC3T3-E1 mouse pre-osteoblast cells. In general, Cu-doped TiO₂ nanotubes presented an almost total antimicrobial action, while Zn doped samples had a lower, but still significant antibacterial effect. However, a highly cytotoxic effect against MC3T3-E1 cells was observed on all anodized samples due to the release of vanadium from the alloy. In spite of this, the surface modification reported in this work can be a valid solution for existing commercially available orthopedic implants, considering that similar solutions were already studied in <i>in vivo</i> assays.