Hydrogen could be alternative energy carrier in the future as well as source for chemical and fuel synthesis due to its high energy content, environmentally friendly technology and zero carbon emissions. In particular, conversion of organic substrates to hydrogen via dark fermentation process is of great interest. The aim of this study was fermentative hydrogen production using anaerobic mixed culture using different carbon sources (mono and disaccharides) and further optimization by varying a number of operating parameters (pH value, temperature, organic loading, mixing intensity). Among all tested mono- and disaccharides, glucose was shown as the preferred carbon source exhibiting hydrogen yield of 1.44 mol H(2)/mol glucose. Further evaluation of selected operating parameters showed that the highest hydrogen yield (1.55 mol H(2)/mol glucose) was obtained at the initial pH value of 6.4, T=37 °C and organic loading of 5 g/L. The obtained results demonstrate that lower hydrogen yield at all other conditions was associated with redirection of metabolic pathways from butyric and acetic (accompanied by H(2) production) to lactic (simultaneous H(2) production is not mandatory) acid production. These results therefore represent an important foundation for the optimization and industrial-scale production of hydrogen from organic substrates.