Tubulins are the proposed target of clinically relevant anticancer drugs, anthelmintic, and fungicide. β₂-tubulin of the plant pathogen Fusarium graminearum was considered as the target of benzimidazole compounds by homology modeling in our previous work. In this study, α₁-, α₂-, and β₂-tubulin of F. graminearum were produced in Escherichia coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant β₂-tubulin and reduced the maximum fluorescence intensity of 2 μM β₂-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibited the polymerization of α₁-/β₂-tubulins and α₂-/β₂-tubulins 90.9 ± 0.4 and 93.5 ± 0.05%, respectively, in vitro. A similar result appeared with benomyl on the polymerization of α₁-/β₂-tubulins and α₂-/β₂-tubulins at 89.9 ± 0.1% and 92.6 ± 1.2% inhibition ratios, respectively. In addition, thiabendazole inhibited 81.6 ± 1% polymerization of α₁-/β₂-tubulins, whereas it had less effect on α₂-/β₂-tubulin polymerization, with 20.1 ± 1.9% inhibition ratio. However, the three compounds cannot destabilize the polymerized microtubule. To illuminate the issue, mapping the carbendazim binding sites and β/α subunit interface on β/α-tubulin complexes by homology modeling showed that the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin-specific anti-F. graminearum compounds.