Distinctions in hydrolysis and acidogenesis were examined for a series of anaerobic batch reactors inoculated with three different anaerobic mixed cultures (mesophilic, thermophilic and hyperthermophilic anaerobic sludge) and operated at the temperature of inoculum’s origin and additionally at 70 °C. Hyperthermophilic temperatures led to increased hydrolysis rates during the start-up stage but a rapid drop in pH limited the overall hydrolysis efficiency, indicating the importance of pH control to sustain the high reaction rates at higher temperatures. No significant difference (P > 0.05) was observed among hydrolysis efficiencies obtained for different reactors which ranged between 27 ± 3% and 40 ± 14%. The highest fermentation yield of 0.44 g COD of fermentation products/g VSS-CODadded was obtained under thermophilic conditions, followed by mesophilic (0.33 g COD ferm. prod./g VSS-CODadded) and hyperthermophilic conditions (0.05–0.08 g COD ferm. prod./g VSS-CODadded). Fermentative performance was better at mesophilic and thermophilic conditions as indicated by improved production of volatile fatty acids (VFA). VFAs accounted for 60–71% of the solubilised matter at thermophilic and mesophilic conditions. Acetic acid formed the primary VFA (70%) at mesophilic temperatures, while butyric acid was the major VFA at thermophilic (60%) conditions. Hyperthermophilic conditions led to increased production of lactic acid, which comprised up to 32% of the solubilised matter. Overall, the results indicate that different operating temperatures may not significantly affect the substrate degradation efficiency but clearly influence the biotransformation pathways.