Polymer molecules, the main components of plastics, are an emerging pollutants in various environmental compartments (water, air, soil) that may induce several ecotoxicological effects on live organisms. Therefore, understanding how plastic particles interact with bacterial cell membranes is crucial in analysing their associated risks in ecosystems and human microbiota. However, relatively little is known about the interaction between nanoplastics and bacteria. The present work focuses onStaphylococcus aureusandKlebsiella pneumoniae, representing the Gram-positive and Gram-negative bacteria respectively, exposed to 100 nm diameter polystyrene nanoparticles (PS NPs). The nanoparticles attach to the cells’ membranes of both bacteria, changing their electrical charge, but without the effect of killing the cells. PS NPs caused a change in zeta potential values (both species of bacterial strains), dependent on particle concentration, pH, as well as on exposure time of bacteria to them. Through the application of AFM and FTIR techniques, the presence of PS NPs on bacterial surfaces was detected, suggesting the affinity of the particles to bacterial components, but without any changes in the morphology of the tested bacteria. The zeta potential can be more widely used in the study of interactions between nanostructures and cells.