In this study, the effects of 5-hydromethylfurfural (5-HMF), levulinic acid, and formic acid on fermentative H-2 production have been investigated. The major byproducts of physicochemical pretreatment of lignocellulosic or algal biomass inhibited H-2 production from glucose and galactose. The half maximal inhibitory concentrations (IC50) of 5-HMF on glucose, levulinic acid on glucose, formic acid on glucose, levulinic acid on galactose and formic acid on galactose were 0.59, 1.55, 12.50, 1.33 and 2.99 g/L, respectively. The pretreatment byproducts levels should be mitigated by controlling the severity of a physicochemical treatment or an additional selective removal process prior to H-2 fermentation. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.