Abstract Oil Palm mesocarp fibre (OPMF), is a byproduct from palm oil production industries, the waste was utilized as a substrate for biogas production. The main barrier in using this type of biomass is its complexity in biodegradation. OPMF was biological pretreated using oyster mushroom for breaking the complex structure so as to expose the lignin to microbial activities and improve the efficiency of anaerobic digestion, and it was subjected to anaerobic digestion with cattle manure (CM) and seeded with Palm oil mill effluent (POME). The biological pretreatment decrease the hemicellulose, cellulose and lignin to 10.91%, 8.96% and 10.63% respectively, lower than the initial lignocellulose content. Laboratory experiment was carried out in a single stage 10 L bioreactor. The reactors were named R1, R2, R3, and R4 at different mixture conditions with organic loading of 1.62 and 2.11 g VSadded L−1 d−1 at HRT of 10 days. R1 produced highest cumulative biogas of 29.16% which was higher than R2, 39.8% higher than R3, and 64.13% higher than R4. This result indicates that R1 which is a mixture of pretreated OPMF with CM seeded with POME produced the best result during the anaerobic digestion process. Thus, anaerobic digestion of pretreated OPMF with CM inoculated with POME shows a better potential for biogas production. Is a good source of biogas production and waste reduction in the palm oil mill industry.