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In this study, the influence of nanometer scale roughness on bacterial adhesion and subsequent biofilm formation has been evaluated using spatially organized microtopographic surface patterns for four major opportunistic pathogens of the genus Staphylococcus ( S. epidermidis and S. aureus ) responsible for associated-biofilm infections on biomedical devices. The results presented demonstrated that regardless of the strain employed the initial adhesion events to these surfaces are directed by cell-surface contact points maximisation and thus, bacterial cells actively choose their position to settle based on that principle. Accordingly, bacterial cells were found to preferably adhere to the square corners and convex walls of recessed surface features rather than the flat or concave walls of equal protruding features. This finding reveals, for the first time, that the particular shape of the surfaces features employed potentially determined the initial location of the adhering cells on textured surfaces. It was further shown that all surfaces patterns investigated produce a significant reduction in bacterial adhesion (40–95%) and biofilm formation (22–58%). This important observation could not be related to physical constrains or increased solid surface hydrophobicity, as previously suggested by other authors using engineered topographies with microscale surface roughness. It is evident that other causes, such as nanoscale surface roughness-induced interaction energies, might be controlling the process of bacterial adhesion and biofilm formation on surfaces with well-defined nanoscale topography. [ABSTRACT FROM AUTHOR]