In order to evaluate the inhibitory effect of eugenol on the biofilm of Bacillus amyloliquefaciens DY1a and the effect of eugenol on the adhesion and aggregation ability of the interface in the early stage of biofilm formation, the effects of eugenol at different mass concentrations on the formation of biofilms, the surface microstructure of biofilms, the number of viable bacteria in biofilms and the content of polysaccharide and protein in biofilm matrix were analyzed, and the scavenging ability of mature biofilms was evaluated. The effect of eugenol on the adhesion and aggregation ability of biofilms in the early stage of biofilm formation was comprehensively analyzed by bacterial motility experiment, cell surface hydrophobicity, cell surface Zeta potential and cell self-aggregation ability. The results showed that the minimum biofilm inhibitory concentration (MBIC) of eugenol against B. amyloliquefaciens was 1.500mg/mL, and the eradication rate of eugenol against mature biofilm was 28.85%. The biofilm formed at the air-liquid interface of eugenol medium supplemented with 1/2 MBIC and 1/4 MBIC was thinner and smoother, and the number of viable bacteria in the biofilm was significantly reduced. Moreover, eugenol was capable of suppressing bacterial swimming (22.16% to 100.00%), swarming motility (43.86% to 97.50%). And cell surface hydrophobicity, negative cell surface Zeta charge, and auto-aggregation were also decreased. In addition, eugenol significantly inhibited exopolysaccharides and protein synthesis of bacteria. Therefore, the antibiofilm mechanism of eugenol against B. amyloliquefaciens DY1a was associated with inhibitory of bacterial motility, changes of cell surface hydrophobicity and auto-aggregation, thus interfering with the adhesion ability of early bacteria at the film forming interface, and inhibiting the synthesis and secretion of extracellular polymeric substance components to delay the growth and maturation of biofilm. In conclusion, eugenol could be used as a potential antibiofilm agent for control biofilm formation of B. amyloliquefaciens at the air-liquid interface.