Electricity generation from wheat straw hydrolysate and the microbial ecology of electricity producing microbial communities developed in two chamber microbial fuel cells (MFCs) were investigated. Power density reached 123 mW/m2 with an initial hydrolysate concentration of 1000 mg-COD/L while Coulombic efficiencies (CEs) ranged from 37.1 to 15.5% corresponding to the initial hydrolysate concentrations from 250 to 2000 mg-COD/L. The suspended bacteria found were different from the bacteria immobilized in the biofilm, and they played different roles in electricity generation from hydrolysate. Bacteria in the biofilm were consortia with sequences similar to Bacteroidetes (40% of sequences), Alphaproteobacteria (20%), Bacilli (20%), Deltaproteobacteria (10%), and Gammaproteobacteria (10%), while suspended consortia were predominated by Bacilli (22.2%). Results from this study can contribute to improve understanding and optimizing the electricity generation in microbial fuel cells.