Studies have been conducted to determine the possibility of using agricultural waste, in particular straw, for the production of biohydrogen. In our work, we used two types of straw hydrolysates (after acidic and enzymatic hydrolysis), pre-activated by steam blasting at steam temperatures of 100, 165, 210 0C. The hydrolysates were diluted with distilled water to a concentration of reducing agents (RR) equal to 1.0, 1.5 and 2.0% of their mass in order to determine the rational concentration. Two cultures of anaerobic microorganisms were tested on 18 samples of hydrolysates: a strain of bacteria of the genus Clostridium Butyricum E.VI .3.2.1 (no. VKPM B-9619), a strain of bacteria of the genus Enterobacter cloacae (no. VKPM B-1980). The fermentation temperature for all samples was set at 37 ± 0.5 0C, pH 5.5 ± 0.1. The released gas accumulated in the gas tank; its samples were taken every 12 hours and analyzed for the volume content of hydrogen. After establishing the rational initial concentration of the substrate, the temperature of steam explosive activation of straw, the type of hydrolysate and the maximum yield of biohydrogen, the cultivation temperature and pH for each culture were further changed in order to determine the rational parameters: for Clostridium Butyricum, the cultivation temperature was set to 32 ±0.5, 37 ±0.5 and 42±0.5 0C for Enterobacter cloacae – 36±0.5, 37±0.5, 38±0.5 0C; the pH for all samples was set in the range of 5...6 in increments of 0.5 ± 0.1). On acidic straw hydrolysates, Clostridium Butyricum demonstrated the best productivity (steam explosion temperature 165 0C) - hydrogen yield was 73 ml/g, on hydrolysates fermented by Enterobacter cloacae (steam explosion temperature 210 0C), hydrogen yield was 50 ml/g. Both cultures are capable of producing the maximum amount of hydrogen at a concentration of reducing substances of 2%. The rational fermentation temperature for Clostridium Butyricum is 37 ± 0.5 0C, for Enterobacter cloacae – 36 ± 0.50 C, for both cultures – pH = 6.