Trifluralin, a synthetic fluorinated dinitroaniline herbicide, was first registered in 1963 and is marketed in a number of names, such as Treflan, Triflurex, Triflusan and others. In 2012, trifluralin was excluded from the State Register of plant protection products and fertilizers permitted for use in the territory of the Republic of Moldova; however, residues of the pesticide are registered in soils of Moldova to the present day. Many methods have been successfully applied for the efficient removal of halogenated pesticides in soils, such as physical adsorption, chemical oxidization and biological degradation. The recent rapid development of the nanotechnology has generated a considerable number of studies dedicated to the usage of iron-based nanoparticles for the degradation of this organic pollutant. The aim of this study was to estimate the sensitivity of microorganisms to magnetite (Fe3O4) nanoparticles and trifluralin, and to evaluate the inhibition activity of these substances. To assess the sensitivity of microorganisms to magnetite nanoparticles and trifluralin in vitro test was carried out. There were used 5 fungal and 5 streptomycetes strains. The solution of Fe3O4 nanoparticles in concentration of 100 mg/L added to medium Czapek had a weak inhibitory effect on 2 fungal strains, no inhibitory effect on 1 strain and stimulated the development of 2 fungi. The inhibitory effect of trifluralin solution, expressed in increasing of the inhibition activity (IA, %), is noticeable in the all studied cultures of fungi. Most sensitive to the inhibitory action of trifluralin was P.viride. After the solution of trifluralin was incubated for one hour with a solution of magnetite nanoparticles, the decreasing of inhibitory activity was observed, and in the case of 2 fungal strains – even a stimulation of the growth of these microorganisms. It should be noted that all of streptomycetes used in the experiment were quite resistant to trifluralin (IA from 7.7% to -5.7%). Trifluralin solution incubated for one hour with a solution of magnetite nanoparticles and added to the medium caused a decrease in the inhibitory activity in all experimental variants. The exception was one streptomycetes strain that showed equal sensitivity to all three solutions. Thus, the solution of magnetite nanoparticles, added to the nutrient medium for fungi and streptomycetes, had not a significant inhibitory effect on fungi and streptomycetes; in contrast, it had a stimulating effect or the lack of any effect. Trifluralin inhibitory effect on fungi and streptomycetes is reduced if the herbicide solution was incubated with the solution of magnetite nanoparticles for one hour prior to addition to the nutrient medium.