Anaerobic digestion (AD) technology can potentially address the gap between energy demand and supply playing a crucial role in the production of sustainable energy from utilization of biogenic waste materials as feedstock. The biogas production from anaerobic digestion is primarily influenced by the chemical compositions and biodegradability of the feedstock. Organosolv-steam explosion offers a constructive approach as a promising pretreatment method for the fractionation of lignocellulosic biomasses delivering high cellulose content.This study showed how synergetic co-digestion serves to overcome the challenges of mono-digestion's low efficiency. Particularly, the study evaluated the digestibility of organosolv-steam pretreated wheat straw (WSOSOL) in mono as well as co-digesting substrate with cheese whey (CW) and brewery spent grains (BSG). The highest methane yield was attained with co-digestion of WSOSOL + CW (338 mL/gVS) representing an enhanced biogas output of 1–1.15 times greater than its mono digestion. An ammonium production was favored under co-digestion strategy accounting for 921 mg/L from WSOSOL + BSG. Metagenomic study was conducted to determine the predominant bacteria and archaea, as well as its variations in their populations and their functional contributions during the AD process. The Firmicutes have been identified as playing a significant role in the hydrolysis process and the initial stages of AD. An enrichment of the most prevalent archaea genera enriched were Methanobacterium, Methanothrix, and Methanosarsina. Reactors digesting simpler substrate CW followed the acetoclastic, while digesting more complex substrates like BSG and WSOSOL followed the hydrogenotrophic pathway for biomethane production. To regulate the process for an enhanced AD process to maximize CH4, a comprehensive understanding of microbial communities is beneficial.