Arbuscular mycorrhizal fungi (AMF) and Bacillus play a crucial role in promoting the growth and defense of exotic plants, and their interaction may further enhance plant invasions. Soil nitrogen level is an important factor that affects the interaction. However, the effect of the interaction on the growth and defensive ability of exotic plants under different nitrogen levels remains unclear. In this study, a pot experiment was conducted using Rhizoglomus intraradices (RI) and Bacillus megaterium (BM), one of the dominant AMF and Bacillus in the rhizosphere of Flaveria bidentis, with three soil nitrogen levels (0, 3.75 and 7.5 g m⁻²) and four inoculation treatments (uninoculated, inoculation with RI, inoculation with BM and co-inoculated with RI and BM). Significant correlations were observed between microbial inoculations and indicators of plant growth and defense across varying soil nitrogen levels. Co-inoculation notably enhanced both plant growth and defense compared to single inoculations, especially under the nitrogen concentration of 3.75 g m⁻². Specifically, compared to single inoculation, co-inoculation increased the biomass of F. bidentis by 8.27% and 16.4%, as well as the flavonoids concentration by 21.89%–30.95% and phenolic acids concentration by 54.22%–60.93%, respectively. These enhancements in growth and defensive compound production likely promote the competitive ability of F. bidentis and its resistance to biotic and abiotic stresses, thereby contributing to its successful invasion.