Two laboratory-scale bioreactors, operated in flow-through mode at fixed pH values, were set up to generate sulfuric acid from elemental sulfur at different temperatures using consortia of sulfur-oxidizing acidophilic prokaryotes. One bioreactor, maintained at 30 °C, was dominated by Acidithiobacillus albertensis when operated at pH 1.0 and by Sulfobacillus thermosulfidooxidans at pH 0.8. Acidibacillus ambivalens (an obligately heterotrophic iron/sulfur-oxidizer) was present at ~10–20% relative abundance at both pH values but Acidithiobacillus caldus was only detected as a minor member of the consortium. In contrast, the other bioreactor, maintained at 50 °C, was populated by At. caldus and the thermophilic archaeon Sulfolobus metallicus at both pH values. Operating both reactors at pH 0.8 was less successful than at pH 1.0, due to the acid-sensitivities of the microbial consortia. However, the total acidities of the sulfuric acid solutions generated could be increased by >90% by increasing sulfate concentrations in the feed liquors, which resulted in most of the acidity generated being in the form of bisulfate (HSO4−) rather than hydronium (H3O+) ions, and obviating, at least partially, the limitations caused by inhibition of the extreme acidophiles to highly elevated concentrations of the latter. Fastest rates of sulfuric acid production under the conditions tested were found when operating the bioreactors at pH 1.0 and 30 °C, with a pH 3.0 influent liquor supplemented with 200 mM magnesium sulfate. This work demonstrated the feasibility of using an empirical flow-through bioreactor to generate a sulfuric acid lixiviant that could be used alone or in tandem with other (microbially-generated) liquors to accelerate the dissolution of minerals and metallic waste materials in bio-processing operations.