We present a nonlinear light scattering method for monitoring, with real-time resolution and membrane specificity, changes in molecular adsorption, and transport at bacterial membranes induced by an antimicrobial compound. Specifically, time-resolved second-harmonic light scattering (SHS) is used to quantify azithromycin-induced changes to bacterial membrane permeability in colloidal suspensions of living Escherichia coli. Variations in membrane properties are monitored through changes in the adsorption and transport rates of malachite green, a hydrophobic cation that gives SHS signal. Regardless of concentration, instantaneous treatment with azithromycin showed no significant changes in membrane permeability. However, 1 h pretreatment with subminimum inhibitory concentrations of azithromycin induced an order-of-magnitude enhancement in the permeability of both the outer membrane and, through facilitation of a new transport mechanism, the cytoplasmic membrane of the bacteria as well. This study illustrates SHS as a novel tool for monitoring antimicrobial-induced changes to membrane properties in living bacteria.