Biosulfidogenesis, the generation of hydrogen sulfide by microorganisms, in acidic (pH 2.5–3.0) bioreactor cultures was investigated using pure cultures of facultatively anaerobic Acidithiobacillus spp. (by sulfur reduction) and an enrichment culture containing acidophilic sulfate-reducing bacteria. Preliminary experiments showed that hydrogen sulfide was produced in anaerobic cultures of Acidithiobacillus ferrooxidans and Acidithiobacillus ferrivorans by sulfur respiration, using hydrogen derived from acid dissolution of zero-valent iron (ZVI) as electron donor. Bioreactors containing strains of these acidophiles, operated at pH 2.5–2.6, were set up to generate H2S that was used to precipitate copper, either off-line (in a separate reactor) or on-line (within the bioreactor itself). When the influent liquor in the At. ferrivorans reactor was changed from copper only to a mixture of copper and zinc, the metals were segregated, with copper precipitating within the bioreactor and zinc remaining in solution. A separate enrichment culture using an acidic, gelatinous microbial mat from a mine drainage stream as an inoculum and ZVI-derived hydrogen as an energy source was able to generate hydrogen sulfide at pH 3 via sulfate reduction. Again, separation of zinc and copper was obtained by selective precipitation of copper sulfide within the reactor vessel. The main SRB identified in the enrichment culture was Desulfosporosinus acidiphilus although other bacteria (but no archaea) with non-ascribed roles within the mixed community were also present. The results demonstrate the potential of biosulfidogenesis in acidic liquors to selectively recover metals from mine drainage waters and related waste streams.