When entering aquatic ecosystems, hydrophobic organic chemicals like the fungicide fludioxonil partition to the sediment compartment where they pose potential risks to benthic invertebrates. To assess the ecological risk for sediment-dwelling invertebrates, nematodes are a suitable organism group, as they are abundantly present and possess key positions in the benthic food web. Therefore, the toxicity of the fungicide fludioxonil to nematodes was assessed in a standardized sediment toxicity test with Caenorhabditis elegans (ISO 10872), as well as in an outdoor sediment-spiked microcosm test system. In the microcosms, effects on the nematode species composition were studied, while exposure concentrations of fludioxonil were monitored in total sediment and pore water. Toxic effects on nematodes were better predicted using concentrations in pore water than total sediment concentrations. In laboratory single species tests, fludioxonil showed considerably lower toxicity in spiked field-collected sediment, compared to artificial ISO-sediments. Applying an assessment factor of 10 to the C. elegans 96-h EC10, a Tier-1 RACNematode of 7.99 mg kg−1 dry artificial sediment (corresponding to 69 μg l−1 in pore water) appeared to be protective for nematode communities in microcosms that showed no response in total abundance and species composition up to 39.9 mg fludioxonil kg−1 dry sediment (corresponding to 392 μg l−1 in pore water).