Using bulk tissue and fatty acid 13C analysis we investigated major trophic pathways from soil microorganisms to microbial consumers to predators in conventional versus organic farming systems planted for the first time with maize. Organic farming led to an increase in microbial biomass in particular that of fungi as indicated by phospholipid fatty acids (PLFAs). Microbial PLFAs reflected the conversion from C3 to C4 plants by a shift in δ13C of 2‰, whereas the isotopic signal in fatty acids (FAs) of Collembola was much more pronounced. In the euedaphic Protaphorura fimata the δ13C values in maize fields exceeded that in C3 (soybean) fields by up to 10‰, indicating a close relationship between diet and vegetation cover. In the epedaphic Orchesella villosaδ13C values shifted by 4‰, suggesting a wider food spectrum including carbon of former C3 crop residues. Differences in δ13C of corresponding FAs in consumers and resources were assessed to assign food web links. P. fimata was suggested as root and fungal feeder in soybean fields, fungal feeder in conventional and leaf consumer in organically managed maize fields. O. villosa likely fed on root and bacteria under soybean, and bacteria and fungi under maize. Comparison of δ13C values in FAs of the cursorial spider Pardosaagrestis and O. villosa implied the latter as important prey species in soybean fields. In contrast, the web-building spider Mangora acalypha showed no predator–prey relationship with Collembola. The determination of δ13C values in trophic biomarker FAs allowed detailed insight into the structure of the decomposer food web and identified diet-shifts in both consumers at the base of the food web and in top predators in organic versus conventional agricultural systems. The results indicate changes in major trophic links and therefore carbon flux through the food web by conversion of conventional into organic farming systems.