This report describes the findings of an investigation of interfacial interactions of bacteria (Pseudomonas aeruginosa) on metal nanoparticles and substrates with different surface properties. In addition to the antibacterial activity of metal nanoparticles, the results of atomic force microscopy (AFM) probing of the interfacial interactions of P. aeruginosa on flat substrates with different surface properties, including a hydrophilic mica and a hydrophobic highly ordered pyrolytic graphite (HOPG), and with different nanoparticle patterns are the focus of this discussion. AFM imaging data revealed that the interaction of the hydrophilic mica surface with P. aeruginosa keeps the cell outer membrane relatively ‘rigid’. The cell outline is largely retained even under the condition of dense packing on the surface. In contrast, the interaction of the hydrophobic HOPG surface renders the cell outer membrane relatively ‘soft’. This soft nature maximises its contact area with the substrate so that the cell outline is altered to fill intercell voids. Demonstration of the viability of creating a nanoparticle-patterned AFM imaging platform in which the assembled nanoparticles inactivate bacteria whereas the bottom substrate promotes bacterial adhesion may find applications in high-throughput screening or assays of bacterial adhesion and antibacterial activity of metal nanoparticles.