AbstractThree white rot fungi (Lenzites betulinus, Trametes orientalis, and Trametes velutina) as well as their respective paired cultures were used to pretreat Populus tomentosa for enhanced lignocellulosic degradation and enzymatic hydrolysis. Hemicellulose and cellulose were slightly degraded, while a maximum lignin degradation of 58% was caused by T. velutina during the 12-week cultivation. was as high as 41%, which was in line with the lignin loss at 2.2 times the control sample. Overall, the monocultures of white-rot fungi exhibited better degradation and saccharification of woody biomass than their co-culture. This can be attributed to the partial removal of lignin and hemicellulose, with an associated increase of cellulose accessibility to enzymes.