The objective of this research was to determine the minimum inhibitory concentration of 5-hydroxymethyl furfural, furfural, and acetic acid on Saccharomyces cerevisiae (NRRL Y-2233) and Zymomonas mobilis subspecies mobilis (NRRL B-4286) in both detoxified hydrolyzed soybean meal and synthetic YM broth spiked with the three compounds. Soybean meal was hydrolyzed with dilute sulfuric acid (0.0, 0.5, 1.25, and 2.0% wt v(-1)) at three temperatures (105, 120, and 135°C) and three durations (15, 30, and 45 min) followed by detoxification with activated carbon. Of all the combinations, only the treatments obtained at 135°C, 2.0% H2SO4, and 45 min and the one at 135°C, 1.25% H2SO4, and 45 min showed inhibition in the growth of the tested microorganisms. Spiked YM broths showed inhibition for the highest levels of inhibitors, either applied individually or in combination.