Abstract: The membranes of infectious fungi and bacteria have emerged as attractive antimicrobial targets due to the relatively rare occurrences of resistance for those drugs active against them. There exists, however, an incomplete understanding of the mechanisms of action for many such membrane-associated small molecule drugs. Using multiple nonlinear microscopy and spectroscopy techniques we are directly probing native small molecule drugs as they interact with the membranes of living bacterial and fungal cells. Specifically, using second harmonic generation spectroscopy we have monitored the interaction of multiple small molecules, including the drug, daptomycin, with living S. aureus and E. faecalis cells. These results have revealed both flip-flop and clustering behavior with subtle changes in structure greatly impacting these dynamics. Further, transient absorption microscopy has allowed for the first ever visualization of the unmodified antifungal drug, amphotericin B, with living S. cerevisiae cells. Our images suggest behavior of this drug that is not consistent with any previously proposed mechanism of action. Overall our work promises new insights into the complex factors governing these important biological systems.