<p>Double refractory gold ores (DRGOs) generally refer to gold ores that contain sulfides encasing gold grains along with carbonaceous matter. Refractory gold ores without carbonaceous matter are conventionally processed by bio-oxidation of the sulfides to liberate and/or expose the gold grains. However, DRGOs have been abandoned for development because the Au(CN)₂⁻ complex easily adsorbs to the carbonaceous matter, resulting in considerable Au recovery loss, which is called “<i>gold robbing</i>.” Several studies have attempted to develop DRGOs, such as through sequential pretreatment by bio-oxidation or chemical leaching followed by enzymatic degradation of the carbonaceous matter because of the different pH value for each step. There have been no reports of one-step pretreatment of DRGOs before cyanidation. This study aims to liberate and/or expose the originally encapsulated Au grains by one-step bio-oxidation treatment of the sulfides and carbonaceous matter in DRGOs using a mixed culture including an iron-oxidizing bacterium (<i>Acidimicrobium ferrooxidans</i>) with/without a sulfur-oxidizing bacterium (<i>Acidithiobacillus caldus</i>) and an acidophilic heterotroph (<i>Alicyclobacillus acidoterrestris</i>) under strongly acidic conditions. Bio-oxidation using the mixed culture completely eliminated the sulfide minerals at low pH, achieved the liberation/exposure of gold grains, and simultaneously modified the carbonaceous matter. In addition, the pretreatment of the DRGO, which is usually a complicated multistep process, was achieved in one step, which is a significant step forward in terms of efficiency.</p> Graphical Abstract <p></p>