The feedstock-specific enzyme systems for saccharification of biofuel feedstocks like switchgrass may potentially provide better enzymatic systems for production of second-generation biofuels. One strategy to develop these enzyme systems could be to harness the microorganisms growing naturally on specific feedstocks. This study presents the isolation and screening of fungal cultures from switchgrass bales for saccharification of ammonia-pretreated switchgrass for subsequent biobutanol production. The best performing fungal isolate during screening was identified through Sanger sequencing of its ITS region to be a unique strain of Trichodenna atroviride and further characterized for production of an enzyme system for saccharification of ammonia pretreated switchgrass. The maximum FPase, CMCase and xylanase activity produced by T. atroviride CUA1 were 0.25 fpu/mL, 0.18 IU/mL and 5.8 IU/mL, respectively. T. atroviride CUA1 also produced considerable amount of B-glucosidase activity. This isolate was used to produce an enzyme system to convert switchgrass to soluble sugars that were then fermented to butanol, ethanol, acetate and butyrate. Glucose was the major product of hydrolysis of ammonia-pretreated switchgrass performed using the enzyme system produced by the isolate. This fungus may be useful for the hydrolysis for the bioenergy crop of switchgrass to overcome this rate-limiting step in the overall conversion of biomass to biofuels. (c) 2014 Elsevier Ltd. All rights reserved.