Nystatin is an antifungal polyene macrolide which is widely applied to treat yeast infections. Nystatin has also been used as a laboratory tool to inhibit endocytic processes in mammalian cells. The interaction of nystatin with model membranes has been studied thoroughly by various spectroscopic methods, making use of its weak fluorescence in the ultraviolet (UV). Studying its interaction with cells would require direct imaging, which, so far, required attachment of a fluorophore to nystatin. Using UV-sensitive microscopy, we show here how to visualize the interaction of nystatin with the plasma membrane (PM) directly. We find that nystatin forms micron-sized aggregates in buffer, and molecular dynamics simulations confirm that nystatin rapidly self-assembles into aggregates in aqueous solution. Using UV-sensitive microscopy, we find that large nystatin aggregates adhere to the surface of Chinese Hamster Ovarian (CHO) cells, causing slow spreading of nystatin fluorescence into the PM. Binding of nystatin to CHO cells does not interfere with cellular uptake or lateral membrane diffusion of the cholesterol analogue TopFluor-cholesterol (TF-Chol). Nystatin binds extensively to the PM of yeast cells as inferred from a strong UV signal in this membrane. Loading a yeast mutant unable to synthesize ergosterol with cholesterol gave much less nystatin membrane staining compared to loading such cells with ergosterol. These results explain the selective fungicidal effect of nystatin by differential interaction of nystatin with yeast membranes containing ergosterol compared to the mammalian cholesterol. Our combined experimental and computational approach provides a toolset for future design of new polyene macrolides.