Pentose and humic acids (HA) are the main components of hydrolysates, the liquid fraction produced during thermohydrolysis of lignocellulosic material. Electricity generation integrated with xylose (typical pentose) degradation as well as the effect of HA on electricity production in microbial fuel cells (MFCs) was examined. Without HA addition the maximum power density increased from 39.5 mW/m2 to 83 mW/m2 when initial xylose concentrations increased from 1.5 to 30 mM, while coulombic efficiency ranged from 13.5% to 52.4% for xylose concentrations of 15 and 0.5 mM, respectively. Compared to controls where HAs were not added, addition of commercial HA resulted in increase of power density and coulombic efficiency, which ranged from 7.5% to 67.4% and 24% to 92.6%, respectively. Digested manure wastewater (DMW) was tested as potential mediator for power generation due to its content of natural HA, and although it could produce higher coulombic efficiency namely 32.2% than the control of 18.3%, showed lower power density which was approx. 57 mW/m2 in comparison to power density of the control which was 69 mW/m2. Presence of commercial HA or DMW in the anode chamber resulted in faster xylose degradation and formation of more oxidized products (acetate and formate) as well as less reduced products (lactate and ethanol) compared to the controls. The reduced power generation in the presence of DMW was attributed to the presence of bacterial inhibitors such as phenolic compounds. Therefore, new feedstocks for MFCs, containing both mediators and substrates, such as lignocellulose hydrolysates should be considered for their applicability in MFCs. Biotechnol. Bioeng. 2008;100: 413–422. © 2008 Wiley Periodicals, Inc.