Microbial biofilms developed on dental implants play a major role in perimplantitis' pathogenesis. Many studies have indicated that surface roughness is the main feature favoring biofilm development in vitro, but its actual influence in vivo has still to be confirmed. In this study, the amount of biofilm formed on differently treated titanium surfaces, showing distinct roughness, has been examined both in vivo and in vitro by Confocal Laser Scanning Microscopy. In vitro studies availed of biofilm developed by Pseudomonas aeruginosa or by salivary bacteria from volunteer donors. In vivo biofilm production was obtained by exposing titanium discs to the oral cavity of healthy volunteers. In vitro experiments showed that P. aeruginosa and, to a lesser extent, salivary bacteria produce more biomass and develop thicker biofilms on laser-treated and sandblasted titanium surfaces with respect to machined ones. In vivo experiments confirmed that bacterial colonization starts on sites of surface unevenness, but failed to disclose biomass differences among biofilms formed on surfaces with different roughness. Our study revealed that biofilm developed in vitro is more easily influenced by surface features than biofilm formed by complex communities in the mouth, where the cooperation of a variety of bacterial species and the presence of a wide range of nutrients and conditions allow bacteria to optimize substrate colonization. Therefore, quantitative differences observed in vitro among surfaces with different characteristics may not be predictive of different colonization rates in vivo.