Cyanidation, a conventional process to extract gold from gold ores, has been used for over 130 years in industrial mining because of the high efficiency and rate of formation of Au(CN)2− and the high recovery efficiency by adsorption of Au(CN)2− on activated carbon. However, carbonaceous refractory gold ores are not targeted for recovering gold because Au(CN)2− is easily adsorbed on carbonaceous matter in the ores, resulting in high recovery loss. In this study, carbonaceous refractory gold ores were subjected to biooxidation at 45 °C using a mixed culture containing iron-oxidizing and sulfur-oxidizing bacteria, following which gold was extracted using thiourea under strongly acidic conditions. The gold extraction rate reached ~100% in 12 h without re-adsorption. Finally, the quantitative recovery of the Au(CS(NH2)2)2+ complex was confirmed by adsorption on strongly cationic exchange resin. The recovery process involved these aspects: (1) biooxidation with the mixed culture containing iron- and sulfur-oxidizing bacteria facilitated sulfide dissolution without the accumulation of elemental sulfur, (2) biooxidation reduced the amount of Fe-containing metal sulfides, which minimized the decomposition of thiourea, and (3) the Au(CS(NH2)2)2+ complex had a low affinity toward carbonaceous matter, different from Au(CN)2−. Since this process does not require roasting to remove carbonaceous materials in pretreatment and does not use cyanide in gold extraction, it is environmentally friendly and must be considered for practical applications in carbonaceous gold ore-producing mines.