How plant species associations affect the rhizosphere microbiota, in comparison to each plant species, has been overlooked. Here we investigated how bi-species plant associations affect total bacterial and N-cycling microbial communities (nitrate reducers and ammonia-oxidizers), as an example of functional communities, at low and high soil N levels. At low N level, the total bacterial abundance in the rhizosphere of the bi-species associations was significantly higher than that of either plant on its own for half of the bi-species associations. For the other associations, the bacterial abundance was not different from the corresponding mono-species cultures. The abundance of several N-cycling guilds was also enhanced by some of the bi-species associations with increases of up to 125% and no negative effects were recorded, which highlights the importance of plant diversity in agro-ecosystems.