Abstract: In order to reduce the dosage and cost of antifungal agents, five green and safe small molecular natural compounds thymol, carvacrol, o-vanillin, citral, and eugenol were selected in this study, and their antifungal effects against Aspergillus flavus (A. flavus) were determined by combination method. Firstly, the minimum inhibitory concentration (MIC) of each compound was detected individually. Then, through chessboard method, citral and eugenol showed a synergistic antifungal effect, and its antifungal mechanism was further studied. The changes of mycelial dry weight of A. flavus under the treatment of citral and eugenol were measured. The effects on cell membrane integrity were detected by propidium iodide (PI) staining, cell content release, relative conductivity and pH value changes. The effect on cell walls was studied by fluorescent white (CW) staining experiment. Finally, the antifungal effect on peanuts was evaluated. The results showed that the MIC of thymol, carvacrol, citral and eugenol were 135, 47, 71 and 43 μg/mL, respectively. Citral and eugenol at the ratio of 1/4+1/4 could inhibit the growth of A. flavus, where the fractional inhibitory concentration index (FIC) was 0.5, indicating that they exerted a synergistic antifungal effect. The dry weight test of mycelia showed that the combination of citral and eugenol (1/4∶1/4) could completely inhibit the growth of mycelia. PI staining experiment showed that citral and eugenol combination treatment destroyed the integrity of A. flavus cell membranes. Compared with the control group, the release of cell content (OD₂₆₀) in 1/16 + 1/16, 1/8 + 1/8 and 1/4 + 1/4 groups increased by about 1.1, 1.2, and 1.5 times respectively. Compared with the control group, at 180 minutes, the extracellular pH value of 1/16+1/16, 1/8+1/8, and 1/4+1/4 decreased by 0.29, 0.32, and 0.49 respectively. Compared with the control group, the relative conductivity of 1/16+1/16, 1/8+1/8 and 1/8+1/8 increased by 5.02%, 18.38% and 33.01% respectively. CW staining experiment showed that the number of septa decreased with the increase of citral and eugenol concentration. When the concentrations of citral and eugenol were 1/16+1/16 and 1/8 + 1/8, there were two septa per picture. When the concentrations of citral and eugenol were 1/8+1/8, the number of septa decreased to one. These results suggested that the synergistic antifungal activity of citral and eugenol against A. flavus was related to the impairment of cell membrane permeability and cell wall integrity. In addition, 1/4 + 1/4 treatment completely inhibited the germination of A. flavus spores on peanuts. Therefore, citral and eugenol treatment showed a promising efficacy in the prevention and control of A. flavus pathogenicity. Meanwhile, such combination treatment method reduced the dosage and cost of natural compounds, and provided a research approach for ensuring food safety.