Fermentative hydrogen production allows the use of renewable biomasses as feedstocks. However, biomass saccharification results not only in carbohydrates, but also in products that can inhibit fermentation. Although biomass hydrolysates contain mixtures of inhibitors, most studies are performed with a single inhibitor. This study evaluates how 5-hydroxymethylfurfural (HMF, 0.60 g/L), levulinic acid (LA, 2.10 g/L), and/or formic acid (FA, 0.80 g/L) mixtures affect two H-2-producing clostridia, Clostridium beijerinckii Br21 and Clostridium acetobutylicum ATCC 824. Fermentation assays with and without (control) the inhibitors helped to calculate the specific H-2-production, substrate consumption, and bacterial cell growth rates for Clostridium beijerinckii Br21 or Clostridium acetobutylicum ATCC 824. HMF + AL, HMF + AF, AL + AF, and HMF + AL + AF mixtures inhibited H-2 production by C. beijerinckii Br21 by 58.7, 60.0, 46.9, and 83.0%, respectively, and by C. acetobutylicum ATCC 824 by 68.1, 71.4, 58.2, and 89.0%, respectively. Clostridium acetobutylicum ATCC 824 metabolized HMF more efficiently. However, organic acids and their combination with HMF inhibited H-2 production by C. beijerinckii Br21 to a lesser extent, which highlighted that this microorganism is robust for H-2 production from biomass hydrolysates. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.