Correctly estimating the trophic fractionation factors (Δ¹⁵N and Δ¹³C) in controlled laboratory conditions is essential for the application of stable isotope analysis in studies on the trophic structure of soil communities. Laboratory experiments usually suggest large ¹⁵N/¹⁴N and small ¹³C/¹²C trophic fractionation, but in field studies litter-dwelling microarthropods and other invertebrates are consistently enriched in ¹³C relative to plant litter. In the present study, we report data from two laboratory experiments investigating both fungi-collembolans and litter-fungi-collembolans systems. In the fungi-collembolans system, Δ¹⁵N and Δ¹³C averaged 1.4 ± 0.1 and 1.0 ± 0.2 ‰, respectively. In microcosms with fungi-inoculated litter, the difference in δ¹⁵N between collembolans and plant litter averaged 1.5 ± 0.2 ‰, confirming the relatively small ¹⁵N/¹⁴N trophic fractionation at the basal level of detrital foodwebs reported in numerous field studies. In full agreement with field observations, the difference in δ¹³C between bulk litter and collembolans in laboratory microcosms averaged 3.6 ± 0.1 ‰ and only little depended on collembolan species identities or the presence of water-soluble compounds in the litter. We conclude that increased δ¹³C values typical of litter-dwelling decomposers are largely determined by an increased ¹³C content in saprotrophic microorganisms.