The flow of benzene carbon along a food chain consisting of bacteria and eukaryotes, including larvae (Diptera: Chironomidae), was evaluated by total lipid fatty acids (TLFAs)-, amino acid- and protein-stable isotope probing (SIP). A coconut-fibre textile, colonized by a benzene-degrading biofilm, was sampled in a system established for the remediation of benzene, toluene, ethylbenzene and xylenes (BTEX)-polluted groundwater and incubated with 12C- and [13C6]-benzene (>99 at.%) in a batch-scale experiment for 2–8 days. After 8 days, Chironomus sp. larvae were added to study carbon flow to higher trophic levels. Gas chromatography-combustion-isotope ratio monitoring mass spectrometry of TLFA showed increased isotope ratios in the 13C-benzene-incubated biofilm. A higher 13C-enrichment was observed in TLFAs, indicative of Gram-negative bacteria than for Gram-positive. Fatty acid indicators of eukaryotes showed significant 13C-incorporation, but to a lower extent than bacterial indicators. Fatty acids extracted from larvae feeding on 13C-biofilm reached an isotopic ratio of 1.55 at.%, illustrating that the larvae feed, to some extent, on labelled biomass. No 13C-incorporation was detectable in larval proteins after their separation by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis and analysis by nano-liquid-chromatography-mass spectrometry. The flow of benzene-derived carbon could be traced in a food web consisting of bacteria and eukaryotes.