Membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, including multidrug resistance (MDR), cellular signaling, and cell-cell communication. Pseudomonas aeruginosa is renowned for its intriguing membrane transport mechanisms, such as the interplay of membrane permeability and extrusion machinery, leading to selective accumulation of specific intracellular substances and MDR. Despite extensive studies, the mechanisms of membrane transport in living microbial cells remain incompletely understood. In this study, we directly measure real-time change of membrane permeability and pore sizes of P. aeruginosa at the nanometer scale using the intrinsic color index (surface plasmon resonance spectra) of silver (Ag) nanoparticles as the nanometer size index probes. The results show that Ag nanoparticles with sizes ranging up to 80 nm are accumulated in living microbial cells, demonstrating that these Ag nanoparticles transport through the inner and outer membrane of the cells. In addition, a greater number of larger intracellular Ag nanoparticles are observed in the cells as chloramphenicol concentration increases, suggesting that chloramphenicol increases membrane permeability and porosity. Furthermore, studies of mutants (nalB-1 and DeltaABM) show that the accumulation rate of intracellular Ag nanoparticles depends on the expression level of the extrusion pump (MexAB-OprM), suggesting that the extrusion pump plays an important role in controlling the accumulation of Ag nanoparticles in living cells. Moreover, the accumulation kinetics measured by Ag nanoparticles are similar to those measured using a small fluorescent molecule (EtBr), eliminating the possibility of steric and size effects of Ag nanoparticle probes. Susceptibility measurements also suggest that a low concentration of Ag nanoparticles (1.3 pM) does not create significant toxicity for the cells, further validating that single Ag nanoparticles (1.3 pM) can be used as biocompatible nanoprobes for the study of membrane transport kinetics in living microbial cells.