The major hurdle to produce bioenergy from rice straw is the low yield of fermentable sugars. This study was carried out to evaluate the effect of pretreatment conditions (rice straw concentration, particle size, hydrolysis time, acid concentration, FeCl3 and enzyme additions) that can yield the maximum sugar monomers for simultaneous production of hydrogen and ethanol by mixed culture fermentation. The results of the evaluation showed that 0.8-1.0 M hydrochloric acid could give the maximum total sugar yield of 52.9%, 2.8 g/L glucose, 14.5 g/L xylose, and 38.6 g/L total reducing sugar from 100 g/L rice straw with particle size 0.15 mm, hydrolysis time 20 min. FeCl3 addition did not enhance the sugar yield but enzymatic hydrolysis increased the reducing sugar yield to 49.8 g/L. High volumetric hydrogen production of 771 mL/L and ethanol production of 1776 mg COD/L was obtained from the pretreated rice straw hydrolyzate. And the total energy from simultaneous hydrogen and ethanol production was highest at 355 J/g polysaccharides. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.