Abstract <p>Conventional sulfuric acid production is tied heavily to the fossil fuel industry, where it is typically obtained from the desulfurization of fuels. Here, the biological production of sulfuric acid by <i>Acidithiobacillus thiooxidans</i> in a reactor setup was optimized using design of experiments methodology. The effects of initial sulfur dosage, phosphate (PO₄³⁻) concentration, and inoculum size were evaluated with respect to sulfate (SO₄²⁻) concentration, optical density, and pH. A significant effect was observed for sulfur dosage; the highest sulfur supplementation, 35 g L⁻¹, yielded the highest SO₄²⁻ concentration and lowest pH after 14 days of cultivation. A mean sulfur-to-SO₄²⁻ conversion rate of 81 ± 9% was determined across all trials conducted with&#xa0;35&#xa0;g&#xa0;L^−&#xa0;1 of sulfur. Among all trials, a maximum SO₄²⁻ molarity of 1.08 M was achieved, with a corresponding pH of 0.18. No significant&#xa0;effects were determined for PO₄³⁻ and inoculum volume. The feasibility of using an industrial by-product as a sulfur source was demonstrated, in addition to establishing a strong correlation between the measured SO₄²⁻ concentration and conductivity of the biological lixiviant.</p> Key points <p>• <i>Sulfur dosage correlated positively with SO</i>_<i>4</i>^<i>2−</i><i> concentration.</i></p> <p>• <i>DoE revealed no significant effect for PO</i>_<i>4</i>^<i>3−</i><i> concentration and inoculum size.</i></p> <p>• <i>Strong correlation between SO</i>_<i>4</i>^<i>2−</i><i> concentration and conductivity was established.</i></p> Graphical Abstract <p></p>