Magnetron sputtering techniques were used to prepare molecularly smooth titanium thin films possessing an average roughness between 0.18 nm and 0.52 nm over 5 μm × 5 μm AFM scanning areas. Films with an average roughness of 0.52 nm or lower were found to restrict the extent of P. aeruginosa cell attachment, with less than 0.5% of all available cells being retained on the surface. The attachment of S. aureus cells was also limited on films with an average surface roughness of 0.52 nm, however they exhibited a remarkable propensity for attachment on the nano-smoother 0.18 nm average surface roughness films, with the attachment density being almost twice as great as that observed on the nano-rougher film. The difference in attachment behaviour can be attributed to the difference in morphology of the rod-shaped P. aeruginosa compared to the spherical S. aureus cells.