Railway transport is a source of pollution to soils and living organisms by e.g. PAHs, PCBs, oil-derived products, pesticides and heavy metals. Soil toxicity evaluation requires chemical analyses, indicating the type and content of particular pollutants, as well as biological analyses, which allow assessing the reaction of organisms to these pollutants. This paper is focused on a multi-aspect evaluation of the degree of toxicity and pollution of soil in selected railway areas from north-eastern Poland by application of numerous biotests and chemical analyses. The soils were sampled on railway tracks from the following railway stations: Białystok Fabryczny, Siemianówka, Hajnówka, Iława Główna and Waliły. The most toxic soils occur on the railway tracks at Białystok Fabryczny and Siemianówka. They had a significant toxic effect on test organisms from various trophic levels. The contents of PAHs, PCBs, heavy metals, oil-derived hydrocarbons and pesticide residues were determined in the examined soils. In all cases the detected pollutants did not exceed the admissible levels. The highest content of oil-derived substances was noted in soils from Białystok Fabryczny and concentrations were moderate in soils from Siemianówka. Although the pollutants determined in soils from railway tracks did not exceed the admissible values, they had a toxic effect on numerous test organisms from different trophic levels. This suggests a synergistic effect of low concentrations (within the admissible levels) of several pollutants together, which resulted in a toxic effect on the organisms. Thus, there is a strong need of not only chemical, but also ecotoxicological analyses during the evaluation of environmental conditions. Based on data obtained from biological and chemical analyses, we concluded that railway transport may pose a hazard to the natural environment to a larger extent that hitherto expected.