The potential effects of engineered metal oxide nanoparticles (MONPs) on bacterial nitrogen fixation are of great concern. Herein, the impact and mechanism of the increasing-used MONPs, including TiO₂, Al₂O₃, and ZnO nanoparticles (TiO₂NP, Al₂O₃NP, and ZnONP, respectively), on nitrogenase activity was studied at the concentrations ranging from 0 to 10 mg L⁻¹ using associative rhizosphere nitrogen-fixing bacteria Pseudomonas stutzeri A1501. Nitrogen fixation capacity was inhibited by MONPs in an increasing degree of TiO₂NP < Al₂O₃NP < ZnONP. Realtime qPCR analysis showed that the expressions of nitrogenase synthesis-related genes, including nifA and nifH, were inhibited significantly when MONPs were added. MONPs could cause the explosion of intracellular ROS, and ROS not only changed the permeability of the membrane but also inhibited the expression of nifA and biofilm formation on the root surface. The repressed nifA gene could inhibit transcriptional activation of nif-specific genes, and ROS reduced the biofilm formation on the root surface which had a negative effect on resisting environmental stress. This study demonstrated that MONPs, including TiO₂NP, Al₂O₃NP, and ZnONP, inhibited bacterial biofilm formation and nitrogen fixation in the rice rhizosphere, which might have a negative effect on the nitrogen cycle in bacteria-rice system.