Lignocellulosic biomass is receiving growing interest as a renewable source of biofuels, chemicals and materials. Lignocellulosic polymers and cellulose nanocrystals (CNCs) present high added-value potential in the nanocomposite field, but some issues have to be solved before large-scale applications. Among them, the interaction between polymers at the nanoscale and the effect of the external parameters on the mechanical properties have to be more precisely investigated. The present study aims at evaluating how the relative humidity affects the reduced Young’s modulus of lignocellulosic films prepared with crystalline cellulose, glucomannan, xylan and lignin and how relative humidity changes their nanoscale adhesion properties with CNCs. Using atomic force microscopy and force volume experiments with CNC-functionalized levers, increasing the relative humidity is shown to decrease the Young’s modulus values of the different films and promote their adhesion forces with CNCs. In particular, CNCs more strongly interact with glucomannan and lignin than xylan, and in the case of lignin, the oxidation of the film promotes strong variations in the adhesion force. Such results allow to better understand the lignocellulosic film properties at the nanoscale, which should lead to an improvement in the production of new highly added-value composites.