The accumulation of water inside wood creates a favorable environment not only for molds, but also for wood-decaying fungi and insects. Therefore, the ability to limit water adsorption and retention is key to the longevity and performance of wood. In this study, the effect of heat-treatment and Cu nanoparticle (CuNP) impregnation on surface contact angle, specific surface area, and hygroscopicity of Masson’s pine wood was examined. Heat-treatment caused thermal degradation of hydroxyl-rich biopolymers, leading to an increase in hydrophobicity; while the resulting breakdown and blockage of the interior cell cavity network caused a decrease in effective surface area. In turn, the hygroscopicity of the heat-treated wood was considerably lower than the untreated wood. Analysis of water adsorption isotherms enabled the differentiation between bound water and free water, where the latter was a prerequisite for mold growth. The research showed that the amount of free water was reduced by both impregnation with CuNP and heat-treatment, but the previously observed antimicrobial activity was shown to rely on the presence of CuNPs as opposed to the reduced free water content. This study presented a detailed methodology for the preparation and analysis of heat-treated, CuNP-impregnated wood, and provided further insight into the mechanism of antimicrobial action of treated woods.