We analysed the prey item composition and microbiome of the African Lake Tanganyika clupeids Stolothrissa tanganicae and Limnothrissa miodon using metabarcoding. We sequenced the mitochondrial COI region of the stomach content for prey analysis and the 16S rRNA region of the hindgut content for microbiome analysis of 120 fish sampled at five locations across Lake Tanganyika. Our research confirmed prey items previously reported in the literature and found a yet unreported prey item, the jellyfish Limnocnida tanganicae. The hindgut harboured 15 bacterial phyla, with Firmicutes and Proteobacteria being the most common. Both species differed in microbiome and diet. The diet of S. tanganicae differentiated on a spatial scale, while the microbiome remained similar. The microbiome of L. miodon differentiated significantly on a spatial scale, while the diet was similar over all locations. Our findings suggest that the Lake Tanganyika clupeids are generalists, with a diet reflecting the local zooplankton community composition. These insights on diet and microbiome provide an excellent reference to monitor the health status of fish stocks.