Abstract In this study, edible filamentous fungi Aspergillus oryzae and Rhizopus oligosporus were co-cultured with fodder yeast Candida utilis using corn thin stillage to produce feed-grade biomass, amylases, and ethanol with the purpose of backset valorization. The co-culture of A. oryzae with C. utilis inoculated after 24 h of pure fungal growth provided the most promising results, yielding 18.6 g L−1 biomass with 41 and 31% of lipid and protein, respectively, with all of the essential amino acids. The microorganisms consumed nearly 34% of the cultivation medium solids, as well as 33% of glycerol and 12% of lactic acid, which allowed them to produce ca. 4.5 g L−1 ethanol and 1.7 U mL−1 amylase. The liquid fraction of the cultivation medium was used as 200 mL L−1 backset for the production of high-solids corn mash. The final ethanol concentrations reached 78.3, 90.6 and 108.2 g L−1 in the control sample (without backset), unfermented backset addition and fermented backset addition, respectively. This suggests that the yeast-fungi co-culture for backset treatment was effective in improving the core production stage in an ethanol-producing plant.