The efficiency of biological nitrate reduction depends on the community composition of microorganisms, the electron donor pool, and the electron mediators participating in the biological reduction process. This study aims at creating an in situ system comprising of denitrifiers, electron donors, and electron mediators to reduce nitrate in surface waters. The ubiquitous periphytic biofilm in waters was employed to promote in situ nitrate reduction in the presence of titanium dioxide (TiO2) nanoparticles (NPs). The nitrate removal rate in the periphytic biofilm and TiO2 NPs system was significantly higher than the control (only periphytic biofilm or TiO2 NPs). TiO2 NPs optimized the community composition of periphytic biofilm for nitrate reduction by increasing the relative abundance of four dominant denitrifying bacteria. Periphytic biofilm showed a substantial increase in extracellular polymeric substance, especially the humic acid and protein content, due to the presence of TiO2 NPs. The synergistic action of humic acid, protein, denitrifying bacteria of the periphytic biofilm, and TiO2 NPs contributed to 80% of the nitrate reduction. The protein and humic acid, acting as electron mediators, facilitated the transfer of exogenous electrons from photoexcited TiO2 NPs to periphytic biofilm containing denitrifiers, which enhanced nitrate reduction in surface waters.