Abstract Anti-biofouling treatment is required in various fields such as biomedical application, construction, civil engineering, and so on. Currently available techniques such as lithography and replica methods have several limitations in application and accessibility. We introduced a simple, biocompatible, and cost-effective anti-biofouling dip-coating method with polyurethane-inorganic (anisotropic montmorillonite and spherical TiO2) hybrid coating agent. Layer thickness of coating was as thin as 5 μm. It was cross-confirmed with thickness gauge and cross-section scanning electron microscopy. Through atomic force microscopy, inorganic nanoparticles were observed to be randomly arrayed with particles partially embedded in the polyurethane network. The calculated surface roughness of inorganic-polyurethane hybrid coating was five times larger than the neat substrate film and three times larger than coating without inorganic nanoparticles. Surface energy of the inorganic-polyurethane film decreased with increasing surface roughness as random pattern of inorganic particle reduced van der Waals interaction. Biofouling efficacy was evaluated by mucin adsorption and consecutive alcian blue assay. Results showed that coated film decreased biofouling 81% compared to bare film.