Compared to antimicrobial agents, anti-adhesive surfaces can reduce bacteria adhesion and biofilm formation in catheters, providing better selectivity, efficiency, and device life span. In this research, novel anionic surface biomaterials were created and tested to reduce microbial adhesion and colonization in medical device coating. Maleic anhydride (MA) was polymerized with 2-HEMA in varying amounts to produce a p(HEMA-co-MA) hydrogel copolymer. Fourier transforms infrared characterization (ATR-FTIR), thermal analysis, scanning electron microscopy with energy-dispersive X-ray spectroscopy, swelling capacity, cytotoxicity evaluation, and mixed biofilm formation ability were used to characterize the copolymer hydrogels. Hydrogels were evaluated by considering the guidance and regulations of ISO and ASTM standards. The polymers were dense, had stable cross-linking between both monomers, were non-toxic to the Human Embryonic Kidney (HEK) 293 cell line, and reduced bacterial biofilm formation statistically significantly. Furthermore, increasing the amount of MA affected TGF-1 gene expression, where the gene expression was significantly elevated, especially at the highest percentage of MA. Furthermore, the high percentage of MA in the polymer improved the new polymer's thermal properties, film flexibility, and swelling capacity. These novel polymers could be promising materials for improving catheter biomaterial properties and modifying the surfaces of designated devices to reduce microbial infections and growth.