In soil food webs, Mesostigmata mites play an important role in regulating populations of other soil organisms. However, their trophic interactions and the factors determining their feeding habits remain poorly understood and are still under-represented in soil food-web studies. Specifically, bottom-up effects of prey diversity and nutritional quality may strongly influence predator fitness and, ultimately, their potential as biological control agents. Several studies have shown that nematodes constitute a key food source for many species within this group of predators. Among soil-dwelling nematophagous Mesostigmata, some species specialize in nematode prey, whereas others consume a broader range of prey but still depend on nematodes for optimal development and reproduction. Most reports refer to the consumption of free-living nematodes (FLN), mainly microbiovores, followed by plant-parasitic nematodes, suggesting that predatory mites may contribute to the transfer of energy and nutrients between detritivore and herbivore chains in soil food webs. FLN, in particular, represent an important source of essential nutrients, including long-chain polyunsaturated fatty acids (PUFAs), which can improve the fitness and reproduction of several Mesostigmata species and may enhance the effectiveness of mite-based biological control. To investigate how different nematode prey influences trophic interactions and predator performance, we combined fatty acid analysis and stable isotope approaches. Feeding experiments involving three species of Mesostigmata revealed that mites generally reflected the lipid profiles of the nematodes they consumed. At the same time, considerable variation in the nutritional quality of different nematode prey was observed, depending on predator species, which may influence their biocontrol efficacy. To further disentangle feeding strategies under semi-natural conditions, dual stable isotope labelling of nematodes (¹³C and ¹5N), coupled with Bayesian mixing models, was applied. Bacteriophage FLN were labelled with ¹³C and plant-parasitic nematodes with ¹5N, allowing direct tracing of prey assimilation by a nematophagous mite species. After short- term interactions, isotopic signatures confirmed predation on both prey types. When both prey were available simultaneously, mites primarily assimilated ¹³C, indicating preferential feeding on FLN while still incorporating smaller amounts of ¹5N from the plant parasite. Together, these results demonstrate that Mesostigmata mites integrate energy from both detritivore and herbivore chains in soil food webs. Predator species differ in their nutritional requirements and responses to different preys, suggesting that maintaining diverse soil communities, including multiple nematode functional groups and organic resources, may be crucial for sustaining predator populations and functional food webs capable of regulating pest populations