Ailanthus altissima is an invasive alien species in Europe. Biological control of this tree species by Verticillium nonalfalfae is a potential alternative control approach. This study investigates host specificity, pathogenicity and transmission of V. nonalfalfae to neighbouring plants with root contact in mini-ecosystems. V. nonalfalfae led to dieback of all inoculated Ailanthus trees. Furthermore, V. nonalfalfae was transmitted to neighbouring Ailanthus trees, causing wilt and dieback, and, in one case, to Quercus petraea, which did not display any disease symptoms. Lastly, V. nonalfalfae could not be detected in the soil, which suggests transmission via root contact.