The lower tier toxicity tests used for risk assessment of plant protection products are conducted with single species, only regarding direct effects of the tested substances. However, it is not clear, if lower tier tests are able to protect in situ soil communities, as these tests are not able to account for direct and indirect effects of chemicals on multi-species systems in natural soil communities. This knowledge gap between single-species tests and field studies can be bridged using model ecosystems (microcosms), which allow for the assessment of direct and indirect effects of the compounds under evaluation. In the present study, single-species toxicity tests and soil-spiked microcosms were used to comparatively investigate the toxicity of the non-systemic fungicide fludioxonil (FDO) on non-target soil organisms, with nematodes being the test organisms of choice. The potential effects of FDO on nematodes were investigated in two different test systems: (i) standardized toxicity tests using Caenorhabditis elegans exposed to FDO-spiked soil (FDO concentrations 50-1207 mg/kg soil dry weight) and (ii) in situ nematode communities sampled from microcosms containing FDO-spiked soil (FDO concentrations 75-600 mg/kg soil dry weight). FDO dose-dependently inhibited the reproduction of C. elegans, with an effect concentration (EC50) of 209.9 mg FDO/kg soil dry weight and a no observed effect concentration (NOEC) of 63.0 mg FDO/kg soil dry weight. In the microcosms, FDO significantly affected trait-based indices, such as the Maturity Index (MI25) and the Enrichment Index (EI), which responded already at FDO concentrations of 14.3 and 62.4 mg/kg dry soil. Overall, this study provides new insights into the impact of the non-systemic fungicide FDO on non-target soil organisms and demonstrates the suitability of nematode-based tools, that allow for a quick and cost-effective lower and higher tier risk assessment of plant protection products.