Palm oil mill effluent (POME) is well known as the potential raw material for biogas production. However, the high hemicellulose and cellulose content (10-14% w/w in dry basis) of the palm fibre in POME are less accessible for biodegradable to sugars, therefore, limiting the carbon source for microorganisms for the biogas production. This study attempted to enhance the hydrolysis step of the biomass by enzymatic pretreatment of POME. The optimum temperature and hydrolysis time using the commercial xylanase (5 unit/mL) were at 50 degrees C for 12 h. Two-steps thermophilic process consisting of POME pretreatment by various concentrations of xylanase (5-20 unit/mL) under the optimum condition followed by biogas production at 60 degrees C for 45 days was carried out. In all cases, the substrate to inoculum volatile solids (VS) ratio and volume ratio were 1:1 and 4:1, respectively. The maximum biomethane (CH4) yield of 914 mL CH4/g VS was obtained from the fermentation of POME hydrolysed by 15 unit/mL of xylanase. However, it was not significantly different from those of the other three concentrations of xylanase (845, 870 and 851 mL CH4/g VS, respectively). They were about 2.5-3 folds higher than that of the control (POME without enzymatic hydrolysis) (297 mL CH4/g VS). Microbial community analysis revealed the presence of Clostridium sp. and Methanocaldococcus sp. as the dominant strains. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.