ABSTRACT Pollutant degradation is present mainly in the surface layer of biofilms, and the surface layer is the most vulnerable to impairment by toxic pollutants. In this work, the effects of nanosized TiO ₂ (n-TiO ₂ ) on the average thicknesses of Bacillus subtilis biofilm and on bacterial attachment on different surfaces were investigated. The binding mechanism of n-TiO ₂ to the cell surface was also probed. The results revealed that n-TiO ₂ caused biofilm dispersal and the thicknesses decreased by 2.0 to 2.6 μm after several hours of exposure. The attachment abilities of bacteria with extracellular polymeric substances (EPS) on hydrophilic surfaces were significantly reduced by 31% and 81% under 10 and 100 mg/liter of n-TiO ₂ , respectively, whereas those of bacteria without EPS were significantly reduced by 43% and 87%, respectively. The attachment abilities of bacteria with and without EPS on hydrophobic surfaces were significantly reduced by 50% and 56%, respectively, under 100 mg/liter of n-TiO ₂ . The results demonstrated that biofilm dispersal can be attributed to the changes in the cell surface structure and the reduction of microbial attachment ability. IMPORTANCE Nanoparticles can penetrate into the outer layer of biofilm in a relatively short period and can bind onto EPS and bacterial surfaces. The current work probed the effects of nanosized TiO ₂ (n-TiO ₂ ) on biofilm thickness, bacterial migration, and surface properties of the cell in the early stage using the surface plasmon resonance waveguide mode. The results demonstrated that n-TiO ₂ decreased the adhesive ability of both cell and EPS and induced bacterial migration and biofilm detachment in several hours. The decreased adhesive ability of microbes and EPS worked against microbial aggregation, reducing the effluent quality in the biological wastewater treatment process.