We studied the adsorption behaviors of organic cations, malachite green (MG), on an anionic dipalmitoylphosphatidylglycerol (DPPG) monolayer at the air–water interface for different thermodynamic phases of the lipid monolayer. Simultaneous monitoring of both the surface pressure and the optical second harmonic generation intensity, combined with the fluorescence microscopy and X-ray reflectivity measurement, gave detailed structural information on the adsorbed MG, showing three distinct types of adsorption behaviors depending on the initial phase of the monolayer. We provided plausible molecular models for the vertical position and orientation of MG in each phase: penetration into the DPPG monolayer with a vertical one direction at a fluid phase; vertically but oppositely paired orientations at a more condensed phase; parallel adsorption at the interface near the lipid headgroup region at a highly packed condensed phase. This result can be usefully expanded for the understanding of the penetration mechanism and conformational change of biological adsorbates at the cellular membrane.