Polystyrene nanoparticles can be chemically synthetized at will or naturally produced by environmental degradation of polystyrene, being considered contaminants in the latter case. Their well-known capacity to induce several eco- and toxicological effects by both in vivo and in vitro studies occurs via cellular uptake and interaction with cell membranes, among others. Considering that the dimensions of most nanoparticles exceed considerably the typical bilayer thickness, the molecular details of nanoparticle-membrane interactions are captivating but still poorly understood. Our study focuses on the interaction of polystyrene nanoparticles with biologically relevant PC:PE:PS (5:3:2) lipid membranes under physiological conditions using quartz crystal microbalance with dissipation monitoring (QCM-D) and planar membrane electrophysiology (PME). Nanoparticles without functionalization (PS) and the functionalized cationic unsaturated amino (PS-NH2) and anionic carboxylated (PS-COOH) with same diameter (∼60 nm) have been tested. QCM-D experiments show that the three types of nanoparticles effectively reach the membrane surface, although in different fashions. Higher and faster depositions were found for PS-NH2 nanoparticles, while PS nanoparticles were found to interact very little with supported lipid membrane. PME recordings show bilayer permeabilization in a similar way than that induced by membrane proteins. Nanoparticle-induced currents show random transitions between different conductive levels of diverse lifetime rather than a progressive disintegration of the membrane. PS-COOH nanoparticles seem to be the least membrane-disruptive, while PS-NH2 nanoparticles turn to have the greatest disruption capacity. Selectivity experiments show currents whose preference for anions or cations is determined by the nanoparticle charge, demonstrating that nanoparticles are an integral part of the generated pores. Overall, our experiments show that nanoparticles do not induce membrane disintegration but probably compromise cell homeostasis in more subtle ways similar to protein ion-channels, suggesting a possible scenario for nanoparticle-induced toxicity.