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This study explored the effect of Fe3O4nanoparticles on Sphingobium yanoikuyaeXLDN2-5, an important environmentally microorganism in carbazole biodegradation process. Microbial cell/Fe3O4nanoparticles biocomposite was constructed by assembling Fe3O4nanoparticles onto the surface of Sphingobium yanoikuyaeXLDN2-5 cells. With the increase in Fe3O4nanoparticles concentration, moderate deterioration in carbazole biodegradation was observed for microbial cell/Fe3O4nanoparticles biocomposites. A concentration-dependent decrease in Sphingobium yanoikuyaeXLDN2-5 viability caused by Fe3O4nanoparticles was observed. Zeta potential measurements suggested that electrostatic repulsion between microbial cell and Fe3O4nanoparticles reduced nanotoxicity at low Fe3O4nanoparticles concentration. The morphology characterization showed the small size of Fe3O4nanoparticle made it possible to penetrate Sphingobium yanoikuyaeXLDN2-5 cell membrane. Furthermore, reactive oxygen species increases, membrane damage, and DNA damage should be responsible for the antibacterial mechanism of Fe3O4nanoparticles at high concentration. This study presented essential knowledge for better understanding of Fe3O4nanoparticles behavior in the environment, and helping to promote safer and more effective application of nanomaterials in nanotechnology-based environmental bioremediation.