Orthopaedic device-related infections are closely linked to biofilm formation on the surfaces of these devices. Several modified titanium (Ti-6Al-4V) surfaces doped with fluorine were studied in order to evaluate the influence of these modifications on biofilm formation by Gram-positive and Gram-negative bacteria as well as a yeast. The biofilm studies were performed according to the standard test method approved by ASTM (Designation: E2196-12) using the Rotating Disk Reactor. Four types of Ti-6Al-4V samples were tested; chemically polished (CP), two types of nanostructures containing fluorine, nanoporous (NP) and nanotubular (NT), and non-nanostructured fluorine containing samples (fluoride barrier layers, FBL). Different species of Gram-positive cocci, (Staphylococcus aureus and epidermidis), Gram-negative rods (Escherichia coli, Pseudomonas aeruginosa), and a yeast (Candida albicans) were studied. For one of the Gram-positive (S. epidermidis) and one of the Gram-negative (E. coli) species a statistically-significant decrease in biofilm accumulation for NP and NT samples was found when compared with the biofilm accumulation on CP samples. The results suggest an effect of the modified materials on the biofilm formation.