Metallurgical dusts are by-products from steel manufacturing. The high iron content of cast house dust (~64%) makes this by-product an interesting iron feedstock alternative. Therefore, its return into the internal steelmaking circuit, specifically in the sinter plant, is a common practice in the steel industry. However, this dust fraction also contains heavy metals, as zinc. As a result of the re-entry of zinc into the process, the zinc concentration in the blast furnace flue gas dust also increases. This prevents the full recirculation of the blast furnace flue gas dust in the steelmaking process despite its relatively high iron content (~35%), thus causing part of the blast furnace flue gas dust to end in the landfill. The goal of this study was to investigate the usage of bacteria, such as the sulfur oxidizing Acidithiobacillus thiooxidans or the iron and sulfur oxidizing Acidithiobacillus ferrooxidans, to leach the undesirable element zinc from the cast house dust while preventing the leaching of iron, by adjusting the sulfur addition and avoiding, at the same time, the accumulation of sulfur in the solid fraction. Experiments proved that a co-culture of A. thiooxidans and A. ferrooxidans can effectively leach zinc from metallurgical dusts, maintaining high iron concentrations in the material. The influence of elemental sulfur on the efficiencies reached was shown, since higher removal efficiencies were achieved with increasing sulfur concentrations. Maximum zinc leaching efficiencies of ~63% (w/w) and an iron enrichment of ~7% (w/w) in the remaining residue were achieved with sulfur concentrations of 15 g/L for cast house gas concentrations of 125 g/L.