The African Lake Tanganyika clupeids play an important role in the lake's ecosystem and have a high regional economic and nutritional value. Using DNA metabarcoding, we analysed the prey item composition and microbiome of these two clupeid species, Stolothrissa tanganicae and Limnothrissa miodon. We sequenced the mitochondrial COI region of the gut content for prey analysis and the 16S rRNA region of the hindgut content for microbiome analysis of 140 fish sampled at five locations across Lake Tanganyika. Our research confirmed previously reported prey items and discovered prey items that were not reported before, including the jellyfish Limnocnida tanganjicae. The hindgut of the fish harboured 15 bacterial phyla, with the most common being Firmicutes and Proteobacteria. The two clupeid species differed in diet, but not in microbiome. Further, the diet of S. tanganicae, but not its microbiome, varied on a spatial scale, whereas the microbiome, but not the diet, of L. miodon showed spatial variation. Our findings suggest that the Lake Tanganyika clupeids are opportunists, with a diet reflecting the local zooplankton community's composition. These results can serve as a useful reference for monitoring the health status of economically important fish stocks.