The majority of human infections are caused by biofilms. The biofilm mode of growth enhances the pathogenicity ofStaphylococcusspp. considerably, because once they adhere, staphylococci embed themselves in a protective, self-produced matrix of extracellular polymeric substances (EPSs). The aim of this study was to investigate the influence of forces of staphylococcal adhesion to different biomaterials onicaA(which regulates the production of EPS matrix components) andcidA(which is associated with cell lysis and extracellular DNA [eDNA] release) gene expression inStaphylococcus aureusbiofilms. Experiments were performed withS. aureusATCC 12600 and its isogenic mutant,S. aureusATCC 12600 Δpbp4, deficient in peptidoglycan cross-linking. Deletion ofpbp4was associated with greater cell wall deformability, while it did not affect the planktonic growth rate, biofilm formation, cell surface hydrophobicity, or zeta potential of the strains. The adhesion forces ofS. aureusATCC 12600 were the strongest on polyethylene (4.9 ± 0.5 nN), intermediate on polymethylmethacrylate (3.1 ± 0.7 nN), and the weakest on stainless steel (1.3 ± 0.2 nN). The production of poly-N-acetylglucosamine, eDNA presence, and expression oficaAgenes decreased with increasing adhesion forces. However, no relation between adhesion forces andcidAexpression was observed. The adhesion forces of the isogenic mutantS. aureusATCC 12600 Δpbp4(deficient in peptidoglycan cross-linking) were much weaker than those of the parent strain and did not show any correlation with the production of poly-N-acetylglucosamine, eDNA presence, or expression of theicaAandcidAgenes. This suggests that adhesion forces modulate the production of the matrix molecule poly-N-acetylglucosamine, eDNA presence, andicaAgene expression by inducing nanoscale cell wall deformation, with cross-linked peptidoglycan layers playing a pivotal role in this adhesion force sensing.