Fractionation of lignocellulosic biomass with solvents (organosolv process) generates a hemicellulose-rich liquor with a high content of phenolics which is particularly toxic. This work addresses the utilization of this stream as a potential carbon source for the production of ligninolytic enzymes (LE). Among six basidiomycetes species, Irpex lacteus and Ganoderma lucidum presented the highest activities of manganese peroxidase (646 ± 122 U L−1) and laccase (1497 ± 161 U L−1), respectively, growing on a medium composed mainly of cellulose fibers, lignin and hemicellulose. The influence of each lignocellulosic fraction on the LE production mechanisms was studied in more detail. The high concentration of phenolic compounds in the hemicellulose-rich stream acted as inducer of LE production, with levels even greater than those of xylose. Acute toxicity tests on Vibrio fischeri revealed a substantial reduction of the toxicity after the fungal treatment (by ca. 12–21 times). The proposed valorization and detoxification of this currently non-exploited and abundant by-product stream is a promising strategy to enhance the industrial feasibility of the organosolv fractionation process.