Abstract Thermal and acid hydrolysates of oil palm empty fruit bunch and other lignocellulosic substrates contain glucose, xylose, and acetic acid as the main components. In using such hydrolysates for photofermentive production of biohydrogen, the gas yield is highly dependent on the composition of the mixed carbon substrate. Batch photofermentation experiments were used to investigate the effect of the composition of the mixed carbon (glucose G, xylose X, and acetic acid A) on growth and hydrogen production by the bacterium Rhodobacter sphaeroides S10. Anaerobic fermentations were carried out at 35 °C under an incident light level of 14.6 W/m2. The mixed carbon composition strongly influenced hydrogen and biomass production. Depending on the composition of the mixed substrate: the cumulative hydrogen volume ranged from 0.99 to 2.33 L H2/L medium; the conversion efficiency ranged from 21% to 45%; and the biomass yield on substrate ranged from 0.28 to 0.47 g DCW/g (G + X + A). Based on the conversion efficiency, the optimal substrate for hydrogen production was a mixture of 5 mM glucose, 18 mM xylose and 7 mM acetic acid. This combination gave a cumulative hydrogen volume of 2.33 L H2/L medium. The hydrogen yield was 3.56 mol H2/mol mixed substrate and the substrate specific hydrogen production rate was 7.26 mL H2/g mixed substrate h. The conversion efficiency and the lag period of hydrogen production were 45% and 13 h, respectively.