Urban road dust (URD) is one of the most important non-point sources of pollution in agglomerations. The aim of this study was to assess the seasonal toxic effects of URD runoff in two regions of Poland. The concentrations of elements in URD and leachate were studied. The impact of pollutants in URD runoff on water organisms was evaluated using Daphtoxkit F and Rotoxkit F (LC50). The acute toxicity tests for crustaceans and rotifers were selected as the response of these taxa reflects the impact on zooplankton, a key component of aquatic ecosystem and the basis of most food webs. The concentrations of elements were found to vary depending on the site, although URD samples collected in Katowice agglomeration (Upper Silesia) had higher values of elements (Mn, Cu, Zn, As) compared to Wrocław (Lower Silesia). The concentrations of Mn, Zn, As, Cr, and Mg in water-soluble fraction of URD were higher in summer and winter in the Upper Silesia region due to rainwater runoff resulting from traffic, industries, post-industrial waste, and the presence of old heating systems. When comparing the content of elements in the water-soluble fraction between seasons, Zn, As, Cr, and Al concentrations were slightly higher in winter. The highest mortality of Daphnia magna and Brachiouns calyciflorus was observed in URD from both agglomerations in winter. However, the mortality is likely due to the concentration of elements or/and the coexistence of an unknown compound or a synergistic effect of the studied elements. This study highlights the alarming seasonal sources of elements in URD runoff, which will directly enter the food chain and affect the entire ecosystem, and human health.