Escherichia coli (E. coli) strains are among the most frequently isolated microorganisms in urinary tract infections able to colonize the surface of urinary catheters and form biofilms. These biofilms are highly resistant to antibiotics and host immune system, resulting in increased morbidity and mortality rates. Strategies to prevent biofilm development, especially via restricting the initial stages of bacteria attachment are therefore urgently needed. Herein, a common urinary catheter material – polydimethylsiloxane (PDMS) – was covalently functionalized with antibacterial aminocellulose nanospheres (ACNSs) using the epoxy/amine grafting chemistry. The PDMS surface was pre-activated with (3-glycidyloxypropyl)-triethoxysilane to introduce epoxy functionalities prior to immobilization of the intact ACNSs via its amino groups. The AC biopolymer was first sonochemically processed into NSs improving by up to 80% its potential to prevent the E. coli biofilm formation on a polystyrene surface. The silicone surface decorated with these NSs demonstrated efficient inhibition of E. coli biofilms, reducing the total biomass when compared with pristine silicone material. Therefore, the functionalization of silicone-based materials with ACNSs shows promise as potential platform for prevention of biofilm-associated infections caused by E. coli.