In this study, freestanding nanobiocomposite films were obtained by the sequential deposition of biopolymer-capped silver nanoparticles (AgNPs) and hyaluronic acid (HA). At first, dispersions ofAgNPs decorated with chitosan (CS) or aminocellulose (AC) were synthesized by applying high intensity ultrasound. These polycationic nanoentities were layer-by-layer assembled with the HA polyanion to generate stable 3D supramolecular constructs, where the biopolymer-capped AgNPs play the dual role of active agent and structural element. SEM images of the assemblies revealed gradual increase of thickness with the number of deposited bilayers. The composites of =50 bilayers were safe to human cells and demonstrated 100% antibacterial activity against Staphylococcus aureus and Escherichia coli. Moreover, the films containing CSAgNPs brought about the total prevention of biofilm formation reducing the cells surface adherence by up to 6 logs. Such nanobiocomposites could serve as an effective barrier to control bacterial growth on injured skin, burns, and chronic wounds.