Investigating and understanding the response of microbes to various surfaces requires a versatile parametrisation of the surface, and multiple assays that captures the complexity of the biofilm structures. Here, Staphylococcus aureus biofilm viability, polysaccharide poly-N-acetylglucosamine, and proteins on the cell surface were analysed with agar plate- and well plate-based biofilm formation assays. Biofilms were grown on a set of nanostructured polymeric surfaces, which were thoroughly characterised for their surface chemistry and topography. Surface hydrophobicity, summit density as well as peak and valley structure were found to influence the microbial viability and exopolysaccharide abundance level in the agar plate assay. In the well plate assay, surface chemical parameters had a lesser influence on the viability, but roughness caused by valley structures increased the viability and decreased the exopolysaccharide expression. Surface proteins relating to pathogenicity were affected by the biofilm formation assay. The abundance profile of these proteins correlated clearly with several roughness parameters, especially fine structure parameters in the agar plate assay and lateral roughness in the well plate assay. These results highlight the necessity of describing the surfaces with a versatile set of different roughness parameters to completely understand what specific features of a surface drive a certain bacterial response.