Double refractory gold ore was sequentially pretreated to oxidize sulfides by thermophilic archaeon Acidianus brierleyi and then to decompose carbonaceous matters using the cell-free spent medium (CFSM) from white-rot fungus Phanerochaete chrysosporium. The pretreatment by A. brierleyi significantly improved the gold recovery from 25% to 77%. Additionally, the crude lignin-degrading enzymes in the CFSM converted the carbonaceous matters into more easily degradable substances, which were removed by alkaline washing, leading to a final gold recovery of 92%. These mineralogical alterations were confirmed by differential thermogravimetric analysis and quantitative evaluation of minerals with scanning electron microscopy. Based on the results, gold grains were mostly liberated after bio-oxidation of sulfides, and in following CFSM treatment, large particles of carbonaceous aluminosilicate were formed from the aggregation of clay minerals, gold grains and with partially decomposed carbonaceous matters acting as binders.