In the present study, the residual toxicity and impact of aged nZVI after a leaching experiment on heavy metal (Pb, Zn) polluted soils was evaluated. No negative effects on physico-chemical soil properties were observed after aged nZVI exposure. The application of nZVI to soil produced a significant increase in Fe availability. The impact on soil biodiversity was assessed by fluorescence in situ hybridization (FISH). A significant effect of nZVI application on microbial structure has been recorded in the Pb-polluted soil nZVI-treated. Soil bacteria molecular response, evaluated by RT-qPCR using exposure biomarkers (pykA, katB) showed a decrease in the cellular activity (pykA) due to enhanced intracellular oxidative stress (katB). Moreover, ecotoxicological standardised test on Caenorhabditis elegans (C. elegans) showed a decrease in the growth endpoint in the Pb-polluted soil, and particularly in the nZVI-treated. A different pattern has been observed in Zn-polluted soils: no changes in soil biodiversity, an increase in biological activity and a significant decrease of Zn toxicity on C. elegans growth were observed after aged nZVI exposure. The results reported indicated that the pollutant and its nZVI interaction should be considered to design soil nanoremediation strategies to immobilise heavy metals. •No negative effects on soil properties were observed after exposure with aged nZVI•The nZVI treatment produced an increase in soil Fe availability•Pb-nZVI soil showed changes in biodiversity, enhanced oxidative stress and Pb toxicity•Increased biological activity and decreased Zn toxicity were observed in Zn-nZVI soil•Impact of aged nZVI exposure mainly depends on the heavy metal contaminant