Biobutanol production from cellulosic feedstock is considered promising and economically feasible. A highly efficient butanol-producing bacterial microflora (containing mainly Clostridial species) was obtained from hydrogen-producing sewage sludge. In this work, two types of agricultural waste (i.e. rice straw and sugarcane bagasse) were alkaline pretreated and then hydrolyzed using a cocktail of cellulases originating from Pseudomonas sp. CL3 and Clostridium sp. TCW1. The hydrolysates were used to produce biobutanol by the isolated mixture culture using either separate hydrolysis and fermentation (SHF) or a combination of SHF with simultaneous saccharification and fermentation (SHF-SSF) processes. In the SHF process, the maximum butanol concentration, productivity, yield and ABE (acetone-butanol-ethanol) ratio from bagasse were 2.29 g/L, 1.00 g/L d, 0.52 mol butanol/mol reducing sugar and 0.12:1:0.06, respectively, and for rice straw were 2.92 g/L, 1.41 g/L d, 0.51 mol butanol/mol reducing sugar and 0.19:1:0.1, respectively. In the SHF-SSF process, the maximum butanol concentration, productivity, and yield for bagasse were 1.95 g/L, 0.61 g/L d and 0.37 mol butanol/mol reducing sugar, respectively, and for rice straw were 2.93 g/L, 0.86 g/L d and 0.49 mol butanol/mol reducing sugar, respectively. This work demonstrated a novel and feasible approach of converting agricultural waste into a valuable biofuel (i.e. butanol). (c) 2012 Elsevier Ltd. All rights reserved.