The bioleaching of the Ag-bearing tetrahedrite mineral originating from the Strieborná vein of the Rožňava mine in eastern Slovakia was investigated. Tetrahedrite from this site is of interest not only because of high Cu (40–46 wt%) and Ag (up to 1%) grades but also because of the potential for Sb recovery. The polymetallic Ag-Cu-Sb-siderite ore sample was dry crushed and milled to a grain size <100 μm. The ground ore was further upgraded by froth flotation and acid leaching, producing a final siderite-free tetrahedrite concentrate. The bioleaching of the concentrate was carried out in an acidic sulfate medium (pH ~ 2) by iron-oxidizing bacteria Acidithiobacillus (At.) ferrooxidans, At. ferrivorans SS3, and Leptospirillum ferriphilum under aerobic conditions at 25 °C. The bioleaching experiments were performed in a 250 mL shaking flask at 190 rpm, with a pulp density of 2%. The period of bioleaching was intentionally extended to 210 days in order to monitor the concentration changes of individual metals and metalloids in the leachate. While the recovery of Cu and Zn increased proportionally with time, the Sb concentration approached 665 mg L−1 (equivalent to 12.7% yield), on approximately the 30th day of bioleaching and subsequently decreased. The concentration of Cu and Zn in the leaching liquor approached 4800 mg L−1 and 115 mg L−1, respectively, which is equivalent to 75% metal yield. The bacterial activity was monitored by online gas analysis using a multichannel respirometric system to monitor O2 consumption during bioleaching.