A new biodegradable nanocomposite was obtained from layer-by-layer (LBL) technique using highly deacetylated chitosan and eucalyptus wood cellulose nanowhiskers (CNWs). Hydrogen bonds and electrostatic interactions between the negatively charged sulfate groups on the whisker surface and the ammonium groups of chitosan were the driving forces for the growth of the multilayered films. The film growth was followed by UV-vis spectroscopy through the maximum value of the absorption band at 194 nm and showed the deposition of 14.7 mg.m(-2) of chitosan polymer in each cycle. Scanning electron microscopy showed high density and homogeneous distribution of CNWs adsorbed on each chitosan layer. Cross-section characterization of the assembled films indicates an average of approximately 7 nm of thickness per bilayer. The results presented in this work indicate that the methodology used can be extended to different biopolymers for the design of new biobased nanocomposites in a wide range of applications such as biomedical and food packaging.