Nanoindentation is an ideal technique to study local mechanical properties of a wide range of materials on the sub-micron scale. It has been widely used to investigate biological materials in the dry state; however, their properties are strongly affected by their moisture content, which until now has not been consistently controlled. In the present study, we developed an experimental set-up for measuring local mechanical properties of materials by nanoindentation in a controlled environment of relative humidity (RH) and temperature. The significance of this new approach in studying biological materials was demonstrated for the secondary cell wall layer (S2) in Spruce wood (Picea abies). The hardness of the cell wall layer decreased from an average of approximately 0.6 GPa at 6% RH down to approximately 0.2 GPa at 79% RH, corresponding to a reduction by a factor of 3. Under the same conditions, the indentation modulus also decreased by about 40%. The newly designed experimental set-up has a strong potential for a variety of applications involving the temperature- and humidity-dependent properties of biological and artificial organic nanocomposites.