Wood-decay fungi cause severe damage to wood by degrading its major structural polymers, thereby reducing the durability and service life of wood products. In this study, the antimicrobial efficacy and biological safety of steam-exploded oak extract was evaluated as a candidate bio-based preservative against wood-decay fungi. Steam-exploded oak was extracted with 50% ethanol, and the resulting extract was applied to pine samples. Treated and untreated samples were exposed to Trametes versicolor (a white rot fungus) and Gloeophyllum trabeum (a brown rot fungus), respectively, for four weeks. After exposure to T. versicolor and G. trabeum, untreated pine samples had significant loss of mass of 41% and 36%, respectively,. In contrast, the extract-impregnated pine samples had marked reductions in loss of mass of 12% and 9%, respectively, demonstrating strong resistance to decay. The cellulase and ligninase of T. versicolor were reduced, and the cellulase activities of G. trabeum were also inhibited. In a cytotoxicity assessment using RAW264.7 macrophages, there was no significant decrease in cell viability, even at an extract concentration of 1,000 ppm. These results suggest that the extract from steam-exploded oak wood has potent antifungal activity against wood-decay fungi, and low cytotoxicity towards mammalian cells. Therefore, this extract can be used as a promising natural and environmentally friendly anti-fungal agent for the preservation of wood.