There are only few studies in the world on ecotoxicity of urban dust as its ability to cause adverse effects to living organisms. The aim of the research is to assess the ecotoxicity of dust by the reaction of higher plants using a new approach, as well as to analyze the differentiation of microbial reaction on dust from recreational, residential and transport functional areas of three Moscow districts. The proposed approach of the urban dust ecotoxicity assessment by the modeling of dust transfer on urban soils proved its consistency and resolved the main methodological difficulty of biotesting – the issue of control choosing. Applying ryegrass (Lollium perenne L.), it was found that the annual volume of dust deposition on soils in Moscow does not perform toxicity, while a three-year volume suppresses plants up to 27% relative to the control. No significant differences between the phytotoxicity of dust in different functional areas was found, and phytotoxicity did not correlate with any of the studied properties of dust (organic carbon content, pH, electrical conductivity, moisture capacity, particle size distribution). In contrast to plants, the parameters of the vital activity of microorganisms in dust revealed differences between functional areas. Basal respiration and microbial dust biomass were maximal in the recreational areas of the city (3.1–7.2 μg C–CO₂g–1 h–1 and 314–435 μg g–1 respectively), decreasing by 27–71% in residential and 76–81% in the transport ones. Significant correlations of basal respiration and microbial biomass with organic carbon content were observed.