The increasing environmental impact of fuels and chemicals from non-renewable fossil-based resources, as well as possible depletion of the latter, has spurred interest in sustainable alternatives. Fuels and chemicals can be produced in a carbon-neutral manner from renewable feedstock via biological processes. Short-and medium-chain fatty acids (SCCA and MCCA, respectively) have potential applications in the food, pharmaceutical, chemical, and biofuel industries. Microbial production of valuable elongated MCCA from SCCA requires an electron donor. The present study investigated the influence of ethanol as an electron donor for the mixed microbial fermentation of SCCA (C2-C5) and MCCA (C6) from brewery spent grains as feedstock. Chain elongation of SCCA to MCCA was evaluated under different ethanol concentrations (3, 6, 9, 12, 15, and 18 g/L) and compared with a non-ethanol control. Acidogenic fermentation successfully converted brewery spent grains to SCCA, reaching 19.66 gCOD/L (15 g/L ethanol supplementation) along with bio-hydrogen production of 41%. Accumulated SCCA were elongated to MCCA in a reverse β oxidation pathway to 9.1 gCOD/L of caproic acid (9 g/L ethanol). Ethanol consumption displayed a good correlation with MCCA formation, confirming the chain elongation capability of mixed cultures. Volatile solids were reduced by more than 70%. Continuous hydrolysis of the substrate with the release of sugars points to the beneficial role of mixed culture fermentation for the production of renewable fuels and chemicals.