Trophic interactions in cryptic belowground systems are difficult to assess, either experimentally or by direct observation. We used lipid analysis to determine feeding strategies in Collembola raised on various bacterial, fungal, plant, or nematode diets. Dietary fatty acids (FAs) were conserved and transferred through the trophic cascade into the neutral lipids of consumers. The presence of vaccenic type FAs (ω7 family) was indicative of a bacterial diet in general. More specifically, methyl‐branched (iso, anteiso) and cyclic forms of FAs were markers for consumption of gram‐positive and gram‐negative bacteria, respectively. Fungal‐feeding Collembola comprised a higher proportion of linoleic acid, whereas the profile of plant feeders showed an increased amount of oleic acid. The FA 20:1 ω9 was only present in Collembola with nematodes as prey. Based on the assigned marker FAs, field populations of Collembola in three deciduous forest stands were ascribed to feeding guilds (i.e., fungivores, bacterivores, herbivores, predators). In conclusion, FA biomarkers provide a high‐resolution method to define feeding strategies of decomposer invertebrates and to determine their diets in situ. Lipid analysis has considerable potential as a new tool in soil food web studies.

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