In the last few years, chitosan-based coatings have been proposed as antibacterial surfaces for biomedical devices in order to prevent nosocomial infections. In that sense, this work reports the optimized synthesis of hyaluronan/chitosan (HA/CHI) nanofilms assembled layer-by-layer in order to maximize the antibacterial effect for two important human pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa. In this assembly, HA forms a soft, highly hydrated, and nontoxic film, whereas CHI shows the antimicrobial characteristics. Our HA/CHI nanofilm synthesis optimization was based on changing pH values of the biopolymer stem-solutions and the consequent variation of their ionization degree. Furthermore, the surface density of primary amino groups, which are related to the antibacterial effect, was also enhanced by increasing the number of HA/CHI bilayers. The antibacterial effect of HA/CHI nanofilms was evaluated by the spread plate counting method for both bacteria. These results were correlated with the morphology of nanofilms (characterized using SEM and AFM), as well as with their chemical properties studied by UV–vis, Kelvin Probe Force microscopy and XPS spectroscopy.