In clinical settings, the intensive use of antibiotics, particularly in intensive care settings, leads to a significant increase in the number of bacterial species that are resistant to treatment. In this context, biofilm is a crucial virulence factor that enable bacteria to persist within the host, often resulting in the need for extensive antibiotic treatment. Staphylococcus epidermidis, a notable nosocomial pathogen, poses a risk to vulnerable patients due to its ability to form biofilms on indwelling medical devices and its high resistance to antibiotic therapy. For this purpose, investigating alternative strategies that target the virulence of pathogens could offer a promising alternative strategy. In this study, we analyzed innovative polymeric materials, such as polysaccharide-based nanohydrogels, for their potential application contrasting S. epidermidis monospecies biofilm on the surfaces of materials most employed in medical devices. These nanohydrogels were found to be effective in eradicating the biofilm matrix and preventing bacterial adhesion. Additionally, the treatment with hyaluronan-based nanohydrogels altered the surface protein profile of S. epidermidis, leading to the disappearance of AtlE, the primary autolysin involved in biofilm formation, suggesting a potential mechanism of action for these nanogels.