Bacterial adhesion to surfaces is accompanied by cell wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Nanoscale cell wall deformation upon adhesion is difficult to measure, except for Delta pbp4 mutants, deficient in peptidoglycan cross-linking. This work explores surface enhanced fluorescence to measure the cell wall deformation of Staphylococci adhering on gold surfaces. Adhesion-related fluorescence enhancement depends on the distance of the bacteria from the surface and the residence-time of the adhering bacteria. A model is forwarded based on the adhesion-related fluorescence enhancement of green-fluorescent microspheres, through which the distance to the surface and cell wall deformation of adhering bacteria can be calculated from their residence-time dependent adhesion-related fluorescence enhancement. The distances between adhering bacteria and a surface, including compression of their extracellular polymeric substance (EPS)-layer, decrease up to 60 min after adhesion, followed by cell wall deformation. Cell wall deformation is independent of the integrity of the EPS-layer and proceeds fastest for a Delta pbp4 strain.