Purpose: Nanoparticles (NPs) have been considered to improve phosphorus (P) availability and activation of soil P in agroecosystems. However, the effects of NPs addition on soil P fractions, microbial characteristics, and plant growth are not well-understood. This study aims to investigate the influences of titanium dioxide (TiO2NPs) and iron oxide (Fe3O4NPs) addition on soil P fractions, microbial characteristics, and plant growth of oilseed rape (Brassica napus L.).Materials and methods: Pot experiment was conducted in 2020 and 2021 years. The exposure of TiO2NPs/Fe3O4NPs (1000 mg kg−1 dry soil) to oilseed rape cultivated was investigated in two contracting calcareous soils (i.e., vegetable field (VF) and cotton field (CF)) for 86 days. Soil pH, Olsen-P, available-Ti/Fe, and Fe-oxides were determined. Different P fractions (CaCl2-P, Citrate-P, Enzyme-P, and HCl-P) were tested by biologically based P fractionation method (BBP). Soil microbial biomass phosphorous (MBP) and alkaline phosphatase activity (ALP) were analyzed. The numbers of bacteria and fungi count were measured by flow cytometry method. Plant biomass and total P uptake were examined in the TiO2NPs/Fe3O4NPs treatments.Results and discussion: Compared with the CK treatment, soil pH was decreased by 12.0–18.0% in the TiO2NPs- and Fe3O4NPs-added treatments in both the VF and CF soils. In contrast, soil Olsen-P was increased by 12.0–19.0%, respectively, implying that TiO2NPs/Fe3O4NPs addition improved soil P availability. The addition of TiO2NPs/Fe3O4NPs significantly affected different soil P fractions. For example, the TiO2NPs/Fe3O4NPs treatments increased CaCl2-P and Citrate-P while decreased Enzyme-P content, indicating that a great portion of soil Enzyme-P was transformed into CaCl2-P and Citrate-P in the TiO2NPs/Fe3O4NPs-treated soils. However, TiO2NPs/Fe3O4NPs addition had no significant influences on HCl-P. Soil microbial biomass phosphorus (MBP), ALP activity, and available-Ti/Fe contents were almost unaffected by TiO2NPs/Fe3O4NPs addition. In addition, the addition of TiO2NPs/Fe3O4NPs had no influence on the numbers of soil bacteria and fungi and plant biomass and total P uptake of oilseed rape.Conclusions: This study demonstrated that the addition of TiO2NPs/Fe3O4NPs in calcareous soils improved soil P availability, and promoted insoluble P transformed to labile-P (CaCl2-P and Citrate-P). However, TiO2NPs/Fe3O4NPs addition at dose of 1000 mg kg−1 dry soil had no toxic effect on oilseed rape (Brassica napus L.). The proper application dosage should be further explored to activate soil P and promote crop growth. Our results provide theoretical basis for the effects of nanoparticles addition on soil P activation, microbial characteristics, and plant growth of Brassica napus L.