Fish play an important role in the transport of energy through tropical food webs. This project proposes a novel method, based on next-generation sequencing, to reconstruct feeding links by examining DNA of gut bacteria. Paired isotope and 16S rRNA gut bacteria sequence data were generated for 61 fish of 10 species, with omnivorous, herbivorous and carnivorous diets, collected from a billabong in Kakadu National Park. This area is of significant social and environmental value for indigenous and non-indigenous peoples. A stepwise distance-based linear model procedure was then used to determine the variation in gut bacteria community composition explained by isotope signatures of basal food sources. Diversity of gut bacteria phyla was higher in fish with omnivorous and herbivorous diets than fish with carnivorous diets. However, bacteria from two phyla (proteobacteria and firmicutes) represented over 50% of the total richness within each fish trophic guild. Stable isotope source data explained ~ 25% of overall gut bacteria community structure, with savanna and planktonic sources explaining most variation. The results confirm previous studies emphasising the role of diet in shaping host gut bacteria. However, the approach could be improved through greater replication of each guild and species, analysis of connectivity between sites and examination of patterns at higher levels of taxonomic resolution. A critical next step will be to incorporate data from another 20 sites that were sampled, to test if bacterial species, functional guilds and communities can be traced through the aquatic food chain.