Magnetite (Fe3O4) nanoparticles (MNP) are widely discussed and suggested as means of environmental remediation, including in cases of soil pollution by persistent organic pollutants and heavy metals. The purpose of this work was to estimate MNP’s potential remediation efficiency and environmental risk by investigating their impacts on the total microbial biomass and its activity in different soils treated with different rates of a persistent organic pollutant (trifluralin – a herbicide banned both in the EU and the Republic of Moldova). MNP were prepared according to the chemical co-precipitation method using iron (II) sulfate and iron (III) chloride in the presence of poly-N-vinylpyrrolidone. The resulting MNP were characterized by X-ray powder diffraction analysis and the scanning electron microscopy. The studied MNP had the size of 20–25 nm and were introduced into soil in the form of crystal powder mixed with talc. The results demonstrated that the introduction of MNP into soil may have both negative and positive impacts on the microbial activity in the relatively unpolluted soils depending on soil type and MNP rates. Fig. 1. The soil microbial biomass (SMB) and the metabolic quotient (qCO2) in a chernozem (A) and greyzem (B) soils depending on the application rates of magnetite nanoparticles (MNP). It was also found that MNP may considerably decrease the toxic impacts on the soil microbial biomass (SMB) from different rates of trifluralin to the point of stimulating the SMB growth (in the presence of this pollutant) above the level in the untreated soil of the pure control (fig. 2). Fig. 2 The impact of magnetite nanoparticles (MNP) on the soil microbial biomass (SMB) in cases of soil treatment with different rates of trifluralin (TF) depending on the vulnerability of the SMB to the TF presence in soil. Conclusions: (1) MNP can have both positive and negative effects on the SMB depending on the application rates and on the soil properties; (2) MNP can be applied only after the environmental risk assessment is done for any given soil; (3) MNP can considerably decrease the toxic impacts on the SMB from different rates of trifluralin to the point of stimulating the SMB growth (in the presence of this pollutant) above the level in the untreated soil (in the pure control).