Atomic force microscopy (AFM) is used to carry out rheology measurements on the nanoscale and to determine the mechanical properties of poly(l-lysine) (PLL)/hyaluronic acid (HA) multilayer films. Storage (G') and loss modulus (G '') of the films are calculated and compared with the values obtained from quartz crystal microbalance with dissipation monitoring measurements (QCM-D). A predominant elastic behavior independently of the applied frequencies (5-100 Hz) is observed for native HA/PLL films consisting of 36 double layer. If the layers are cross-linked, the value of G' increases by 2 orders of magnitude, while the loss modulus becomes negligible, making these films a purely elastic chemical gel. The values of G' and G '' extracted from QCM-D measurements on native films are much higher, due to the different frequency regime of the applied shear stress. However, the viscoelastic ratio from the two methods is the same and proves the elastic dominated response of the multilayer in both frequency regimes.