Optimization of batch pyrite bioleaching with Sulfolobus acidocaldarius was performed using statistical modelling and experimental design. First a screening design was made followed by response surface modelling. The dominating factors identified were pH, pulp density and particle size. The highest batch leaching rate after optimization was 270 mg iron·l−1·h−1 for 6% (w/v) pulp density, pH = 1.5 and particle size <20 μm. This represents a 3.5-fold increase from the leaching rate of 80 mg iron·l−1·h−1 obtained under our standard laboratory conditions.