Ti–6Al–4V joint replacement implants foster uncemented fixation in orthopaedic surgery. However, bacterial colonization competes with host cells and ultimately may produce implant-related difficult-to-treat infections, justifying the efforts to obtain infection-resistant materials. In a previous work, the authors demonstrated the antibacterial properties of anodic fluoride-TiO 2 nanostructured layers on Ti–6Al–4V alloy. In this work, the anodizing bath has been modified in order to grow fluoride-TiO 2 barrier layers (FBL). A bacterial adherence protocol, run with reference and six different clinical strains of Staphylococcus aureus and Staphylococcus epidermidis, showed a statistically significant decrease in the percentage of covered surface (p<0.0001, Kruskal–Wallis test) for FBL specimens when compared with non fluoride-containing specimens, i.e. chemically polished Ti–6Al–4V and F-free TiO 2 barrier layers. The results obtained on the F-barrier layers allowed discrimination between the effects of the presence of fluoride in the layer and the layer nanostructure on bacterial adhesion.