Graphene and its derivatives, such as graphene oxide (GO) and reduced graphene oxide (rGO), have propelled advancements in biosensor research owing to their unique physicochemical and electronic characteristics. To ensure their safe and effective utilization in biological environments, it is crucial to understand how these graphene-based nanomaterials (GNMs) interact with a biological milieu. The present study depicts GNM-induced structural changes in a self-assembled phospholipid monolayer formed at an air-water interface that can be considered to represent one of the leaflets of a cellular membrane. Surface pressure-area isotherm and electrostatic surface potential measurements, along with advanced X-ray scattering techniques, have been utilized in this study. Experimental findings demonstrate a strong interaction between negatively charged GO flakes and a positively charged monolayer, primarily dictated by electrostatic forces. These GO flakes assemble horizontally beneath the head groups of the monolayer. In contrast, rGO flakes permeate the zwitterionic lipid layer through dominant hydrophobic interaction. This organization of GNMs alters the in-plane elasticity of the lipid film, exhibiting a drop in the electrostatic potential of the surface according to the extent of oxygen-containing groups. These results provide a solid groundwork for designing devices and sensors aimed at augmenting the biomedical applications of GNMs.