Titanium dioxide nanoparticle (TiO2 NP) toxicity to the growth of organisms has been gradually clarified; however, its effects on microorganism-mediated phosphorus turnover are poorly understood. To evaluate the influences of TiO2 NPs on phosphorus fractionation and the bacterial community, aquatic microorganisms were exposed to different concentrations of TiO2 NPs with different exposure times (i.e., 0, 10, and 30 days). We observed the adhesion of TiO2 NPs to the cell surfaces of planktonic microbes by using SEM, EDS, and XRD techniques. The addition of TiO2 NPs resulted in a decrease in the total phosphorus of water and an increase in the total phosphorus of sediments. Additionally, elevated TiO2 NPs enhanced the sediment activities of reductases (i.e., dehydrogenase [0.19–2.25 μg/d/g] and catalase [1.06–2.92 μmol/d/g]), and significantly decreased the absolute abundances of phosphorus-cycling-related genes (i.e., gcd [1.78 × 104–9.55 × 105 copies/g], phoD [5.50 × 103–5.49 × 107 copies/g], pstS [4.17 × 102–1.58 × 106 copies/g]), and sediment bacterial diversity. TiO2 NPs could noticeably affect the bacterial community, showing dramatic divergences in relative abundances (e.g., Actinobacteria, Acidobacteria, and Firmicutes), coexistence patterns, and functional redundancies (e.g., translation and transcription). Our results emphasized that the TiO2 NP amount—rather than the exposure time—showed significant effects on phosphorus fractions, enzyme activity, phosphorus-cycling-related gene abundance, and bacterial diversity, whereas the exposure time exhibited a greater influence on the composition and function of the sediment bacterial community than the TiO2 NP amount. Our findings clarify the responses of phosphorus fractions and the bacterial community to TiO2 NP exposure in the water–sediment ecosystem and highlight potential environmental risks of the migration of untreated TiO2 NPs to aquatic ecosystems.