Agro-industrial lignocellulosic waste can be bioconverted to fungal biomass and then utilized as a high nutritional value food alternative for ruminants, with potential for ruminal methane (CH4) mitigation. This study was carried out to evaluate the changes in the enzymatic expression and nutritional value of palm kernel cake fermented with Pleurotus ostreatus (FPKC), as well as the differences in the in vitro fermentation parameters and methane production in diets based on tropical forages with the inclusion of 20% FPKC. The highest activity of laccases and cellulases occurred on day 13 with values of 0.75 ± 0.09 U/g and 266.7 ± 20.8 U/g; respectively. The biological pretreatment decreased the contents of (p< 0.05) neutral detergent fiber (NDF), acid detergent fiber (ADF) and lignin (LIG) by 29%, 20.5% and 46.6% respectively, while those of crude protein (CP) increased by 69.65%. The inclusion of FPKC resulted in increases (p