In an effort to achieve sustainable development goals, a reevaluation of the materials used in wooden buildings must be done, including the preservatives used to treat the materials. Since typical wood preservatives use toxic heavy metals, their handling and use can contaminate the environment. Therefore, substances such as lignin-derived components have been investigated as bio-based preservatives. Organosolv treatment is a promising technique for separating components of lignocellulosic biomass, which enables the utilization of each component. The present report describes components of lignocellulose with antifungal effects that were recovered after organosolv treatment using water and 1-butanol solvent at 473 K for 2 h, followed by simple solvent fractionation. The organosolv lignin was divided into three fractions: n-hexane soluble, ethyl acetate soluble, and ethyl acetate insoluble, yielding 23 wt%, 52 wt% and 13 wt%, respectively. Antifungal activity was determined using an agar plate method. White rot fungi (Trametes versicolor) was dispersed on the agar plate with a cellulose disc containing each lignin-derived fraction obtained from Japanese cedar. Results showed inhibition of fungal growth over the cellulose disc containing the n-hexane soluble fraction. To examine the effect in greater detail, the chemical structure of the n-hexane-soluble fraction on the antifungal activity was investigated. The content of phenolic hydroxyl group in n-hexane-soluble fraction was the highest (4.6 mmol/g), and the results from the chemical modification suggested that the functional group was required for antifungal action. In addition, the n-hexane-soluble fraction imparted some water resistance. The procedures used for cedar as a feedstock were applied to another type of biomass—bagasse—and its fractions showed antifungal activity similar to those of Japanese cedar.