Soybean (Glycine max L.) grain samples with moisture content (MC) varying between 11.3 and 17% (equilibrium relative humidity, respectively, 66.5, 73.1, 79.7 and 85.4% at 25°C) and inoculated with conidia of Aspergillus glaucus were stored for 135 days at 25 ± 2°C, and fumigated with mustard essential oil (MEO) at the rate of 50 or 100 µL/L. Deterioration of the samples was assessed by estimating the increase of colony forming units (CFU) and free fatty acid (FFA) accumulation. Although the values of these indicators increased with increasing MC and storage period, with or without fumigation, the rate of increase was very slow in the fumigated samples. In grains at 17% MC, the CFU number was 10³-fold and FFA content was 6-fold higher in non-fumigated grains compared with the grains fumigated with 100 µL MEO/L, after 4.5 months of storage. No visibly moulded grains were found in the fumigated samples, while such grains were abundant in non-fumigated samples at 17 or 14.8% MC after 45 or 75 days of storage, respectively. The retardation of fungal growth and FFA accumulation appeared to be due to reduced Aspergillus inoculum on the grain surface. There was a significant positive correlation between CFU counts and FFA content. The results of this study suggest that MEO can be used as an eco-friendly and safe antifungal fumigant to prolong storage life of soybeans.