Biofouling remains a persistent issue in biomedical applications such as tissue engineering, wound healing, and the use of medical devices. Developing antifouling coatings that can perform reliably on both hydrophilic and hydrophobic surfaces is a significant challenge. In this work, amphiphilic random copolymers composed of poly(ethylene glycol) methyl ether methacrylate and 2-acetoacetoxy ethyl methacrylate [P(OEGMA-co-AEMA)] were synthesized through controlled radical polymerization. These copolymers were deposited onto substrates using the Langmuir-Blodgett technique, enabling precise control over film formation at the air-water interface. Structural analysis using atomic force microscopy, ellipsometry, and adhesion force mapping showed that the copolymers self-assembled into nanostructured domains due to phase segregation between hydrophilic and hydrophobic segments. The orientation of these domains was influenced by the underlying substrate: OEGMA segments aligned toward hydrophilic surfaces, while AEMA segments oriented toward hydrophobic ones. This substrate-responsive organization led to uniform film coverage and distinct surface morphologies tailored to the surface chemistry. Bacterial adhesion assays demonstrated that the coatings significantly reduced fouling, achieving up to 80 % reduction in bacterial attachment on both types of substrates. The ability of these nanostructured films to adapt their configuration based on surface properties, while maintaining effective antifouling behavior, underscores their potential as broadly applicable surface coatings. These results support the development of versatile, high-performance antifouling materials suited to a wide range of biomedical environments.