Lignocellulose biodegradation is limited because of its recalcitrant structure particularly when polluted by toxic and carcinogenic compounds such as creosote oil (CRO). As far as we know, this might be the first report that explores the biodegradation of creosote treated wood (CTW) to serve biomethane production. Two novel CTW-degrading microbial consortia, designated as CTW-1 and CTW-2, were screened and constructed to enhance methane production from CRO-treated pine sawdust. After 12 days of biological pretreatment by CTW-1 and CTW-2, a significant reduction in lignocellulosic content of CTW was recorded; estimated as 49 and 43%, respectively. More than 64 and 91% of cumulative biogas and methane yields were obtained from biodegraded CTW over control. Ecotoxicity of treated and untreated CTW was compared by Microtox test. The biodegraded CTW hydrolysates showed a toxicity decrease of more than 80%, suggesting the promising role of constructed microbial consortia for biofuel production and bioremediation.