The ‘Enemy of my enemy’ (EE) is a major hypothesis in invasion ecology. It states that a non-native invader ‘accumulates generalist pathogens, which limit competition from indigenous competitors’. Few empirical studies have tested the EE hypothesis in plant invasions, especially on biotic rhizosphere interactions. Here, the EE hypothesis was tested by applying rhizosphere biota from the invasive plant Vincetoxicum rossicum (VIRO) to five co-occurring native plant species, and four native legume species, respectively. Each of the native plant species, and VIRO were grown under controlled conditions for three months, either in presence or absence of soil biota from VIRO invaded and non-invaded soils. Rhizosphere biota from invaded areas had variable effects among native plants (including legumes). It was concluded that the accumulation of rhizosphere enemies that ‘spill’ onto native plants may not be a major factor in the invasive success of VIRO. The EE hypothesis was not supported.