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In this study fungal pretreatment of willow sawdust (WSD) via the white rot fungi Leiotrametes menziesii and Abortiporus biennis was studied and the effect on fractionation of lignocellulosic biomass and biochemical methane potential (BMP), was evaluated. Scanning electron microscopy (SEM) and IR spectroscopy were used to investigate the changes in the structural characteristics of the pretreated WSD. Fungal pretreatment results revealed that A. biennis is more attractive, since it resulted in higher lignin degradation and lower holocellulose uptake. Samples of the 14th and 30th d of cultivation (i.e. the middle and the end of the pretreatment experiment) with both fungi were used for BMP tests and the effect of pretreatment duration was also evaluated. BMP increase by 31 and 43% was obtained due to the cultivation of WSD with A. biennis , for 14 and 30 d, respectively. In addition, combination of biological (after 30 d of cultivation) with alkaline (NaOH 20 g/100 gTS) pretreatment was performed, in order to assess the effect of the chemical agent on biologically pretreated WSD, in terms of lignocellulosic content and BMP. Combination of alkaline with fungal pretreatment led to high lignin degradation for both fungi, while the cellulose and hemicellulose removal efficiencies were higher for combined alkaline and L. menziesii pretreatment. The maximum BMP was observed for the combined alkaline and A. biennis pretreatment and was 12.5 and 50.1% higher than the respective alkaline and fungal pretreatment alone and 115% higher than the respective BMP of raw WSD. [ABSTRACT FROM AUTHOR]