To provide insights into the antifungal effect of 2-ethylhexanol, its effects on Aspergillus flavus spore germination, aflatoxin B₁ (AFB₁) production, cell damage, related gene expression, and control of A. flavus in maize, wheat, and peanuts were evaluated. A. flavus spore germination was completely inhibited by 2-ethylhexanol at 0.17 μL/mL using fumigation method, or at 0.78 μL/mL using liquid mixing method. A. flavus biomass and AFB₁ content were substantially reduced, the cell wall and cell membrane were disrupted by 2-ethylhexanol, causing cell content leakage, reactive oxygen species accumulation, and DNA damage. Furthermore, A. flavus genes related to ergosterol synthesis and aflatoxins global regulator were downregulated by 2-ethylhexanol. The A. flavus population, free fatty acid and AFB₁ content of the three tested grains were notably reduced by 2-ethylhexanol fumigation. These results demonstrated the potential application value of 2-ethylhexanol in the control of A. flavus in food and grains.

Volatile organic compounds (VOCs) produced by Bacillus species exhibit biocontrol activity against fungal pathogens of fruits and vegetables. However, research on the effect of VOCs on Aspergillus flavus in stored grains is limited. This study aimed to investigate the effects of VOCs extracted from the strain R2, which was isolated from unhulled rice and identified as Bacillus paramycoides on A. flavus in vitro and unhulled rice. R2 VOCs effectively inhibited conidial germination and the hyphal growth of A. flavus in vitro. Moreover, R2 VOCs reduced the fungal population, aflatoxin B₁ (AFB₁) levels, and free fatty acid (FFA) value by 90.8%, 67%, and 38.7%, respectively, in unhulled rice. Eighteen R2 VOCs were identified using headspace solid-phase micro-extraction gas chromatography–mass spectrometry, and the individual activity of the VOCs against A. flavus was tested in vitro. Benzaldehyde (Ben) and 3,7-dimethyl-1-octanol (Dmo) showed strong inhibitory activities against A. flavus on PDA plates, with inhibition rates of 100% and 91.2%, respectively, at a concentration of 20 μL/dish. Ben at the concentration of 0.09 mg/mL, Dmo at the concentration of 0.07 mg/mL, or a mixture of both at halved concentrations could reduce the fungal population, AFB₁ levels, and FFA content in unhulled rice. Our findings suggest that R2 VOCs are good alternatives to traditional chemical fumigants for suppressing A. flavus in stored grains. However, further research is necessary to establish the optimal fumigation concentration of these two components in unhulled rice. The impact of their residues on grain quality should be explored through sensory evaluation and nutritional analysis, and their safety to the environment and human body should be evaluated through safety assessment.