Nanopillared surfaces have emerged as a promising strategy to combat bacterial infections on medical devices. However, the mechanisms that underpin nanopillar-induced rupture of the bacterial cell membrane remain speculative. In this study, we have tested three medically relevant poly(ethylene terephthalate) (PET) nanopillared-surfaces with well-defined nanotopographies against both Gram-negative and Gram-positive bacteria. Focused ion beam scanning electron microscopy (FIB-SEM) and contact mechanics analysis were utilised to understand the nanobiophysical response of the bacterial cell envelope to a single nanopillar. Given their importance to bacterial adhesion, the contribution of bacterial surface proteins to nanotopography-mediated cell envelope damage was also investigated. We found that, whilst cell envelope deformation was affected by the nanopillar tip diameter, the nanopillar density affected bacterial metabolic activities. Moreover, three different types of bacterial cell envelope deformation were observed upon contact of bacteria with the nanopillared surfaces. These were attributed to bacterial responses to cell wall stresses resulting from the high intrinsic pressure caused by the engagement of nanopillars by bacterial surface proteins. Such influences of bacterial surface proteins on the antibacterial action of nanopillars have not been previously reported. Our findings will be valuable to the improved design and fabrication of effective antibacterial surfaces.