Identification of membrane sites capable of drug binding is important to understand the interaction of bioactive compounds with biointerfaces such as microbial cellular membranes. As the exact molecular mechanism of action is yet to be fully understood for many drugs, in this study, polygodial, an antimicrobial compound, was incorporated in Langmuir monolayers of selected lipids that served as cell membrane models. Results from pressure-area isotherms, specific vibrational spectroscopy and Brewster angle microscopy revealed the expansion of the lipid monolayers, but with distinctive effects for each lipid in terms of film compressibility, stability, order of alkyl chains, and topographical morphology. These data can be therefore correlated with the possible bioactivity effect of polygodial in lipidic surfaces of mammalian and microbial membranes.