Multilayered films prepared from graphene oxide (GO) subjected to a single oxidation process (1GO) can actuate in response to moisture, whereas those prepared from GO subjected to two oxidation processes (2GO) lose this ability. To elucidate the origin of this difference, the structures and properties of various multilayered films and their contents were analyzed. According to atomic force microscopy images, the lateral size of the GO monolayer in 2GO (2.0 +/- 0.4 mu m) was smaller than that in 1GO (3.2 +/- 0.4 mu m), although this size difference did not affect actuation. Scanning electron microscopy images of the cross sections of both films showed fine multilayered structures and X-ray diffraction measurements showed the moisture sensitive reversible change in the interlayer distances for both films. Both films adsorbed 30 wt% moisture in 60 s with different water contents at the bottom moist sides and top air sides of the films. Nanoindentation experiments showed hardness values (1GO: 156 +/- 67 MPa; 2GO: 189 +/- 97 MPa) and elastic modulus values (1GO: 4.7 +/- 1.7 GPa; 2GO: 5.8 +/- 3.2 GPa) typical of GO, with no substantial difference between the films. On the contrary, the 1GO film bent when subjected to a weight equal to its own weight, whereas the 2GO film did not. Such differences in the macroscopic hardness of GO films can affect their moisture-induced actuation ability.