The aim of this work is to evaluate biohydrogen production from agro-industrial waste-waters and by-products, by combining dark fermentation and microbial electrolysis in a two-step cascade process. Such coupling of both technologies constitutes a technological building block within a concept of environmental biorefinery where sustainable production of renewable energy is expected. Six different wastewaters and industrial by-products coming from cheese, fruit juice, paper, sugar, fruit processing and spirits factories were evaluated for the feasibility of hydrogen production in a two-step process. The overall hydrogen production when coupling dark fermentation and microbial electrolysis was increased up to 13 times when compared to fermentation alone, achieving a maximum overall hydrogen yield of 1608.6 +/- 266.2 mLH(2)/gCOD(consumed) and a maximum of 78.5 +/- 5.7% of COD removal. These results show that dark fermentation coupled with microbial electrolysis is a highly promising option to maximize the conversion of agro-industrial wastewaters and by-products into bio-hydrogen. (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.