Abstract Background Earth’s climate is warming as a result of anthropogenic emissions of greenhouse gases from fossil fuel combustion. Bioenergy, which includes biodiesel, biohydrogen and bioethanol, has emerged as a sustainable alternative fuel source. For this reason, in recent years biodiesel production has become widespread but this industry currently generates a huge amount of glycerol as a by-product, which has become an environmental problem in its own right. A feasible possibility to solve this problem is the use of waste glycerol as a carbon source for microbial transformation into biofuels such as hydrogen and ethanol. For instance, Escherichia coli is a microorganism that can synthesize these compounds under anaerobic conditions. Results In this work an experimental procedure was established for screening E. coli single mutants to identify strains with enhanced ethanol and/or H 2 productions compared to the wild type strain. In an initial screening of 150 single mutants, 12 novel strains ( gnd , tdcE , rpiA nanE , tdcB , deoB , sucB , cpsG , frmA , glgC , fumA and gadB ) were found to provide enhanced yields for at least one of the target products. The mutations, that improve most significantly the parameters evaluated ( gnd and tdcE genes), were combined with other mutations in three engineered E. coli mutant strains in order to further redirect carbon flux towards the desired products. Conclusions This methodology can be a useful tool to disclose the metabolic pathways that are more susceptible to manipulation in order to obtain higher molar yields of hydrogen and ethanol using glycerol as main carbon source in multiple E. coli mutants.