Ester–aldehyde fraction (EAF) is a by-product of ethyl-alcohol-producing companies whose purification requires an expensive process. The results of this study illustrate the environmentally friendly and alternative possibility of using EAF to increase their value as substrate to produce α-ketoglutaric acid (KGA) using different yeasts. It was found that some species of the genera Babjeviella, Diutina, Moesziomyces, Pichia, Saturnispora, Sugiyamaella, Yarrowia and Zygoascus grown under thiamine deficiency accumulate KGA in the medium with an EAF as the sole carbon source. The strain Y. lipolytica VKM Y-2412 was selected as the producer. To reach the maximum production of KGA, the cultivation medium should contain 0.3 µg/L thiamine during cultivation in flasks and 2 µg/L in the fermentor; the concentration of (NH4)2SO4 should range from 3 to 6 g/L; and the optimal concentrations of Zn2+, Fe2+ and Cu2+ ions should be 1.2, 0.6 and 0.05 mg/L, respectively. EAF concentration should not exceed 1.5 g/L in the growth phase and 3 g/L in the KGA synthesis phase. At higher EAF concentrations, acetic acid was accumulated and inhibited yeast growth and KGA production. Under optimal conditions, the producer accumulated 53.8 g/L KGA with a yield (Yp/s) of 0.68 g/g substrate consumed.