Understanding how diversity is partitioned along natural and anthropogenic gradients within ecosystems is important to predict the persistence of species and the ecological functions they provide. Dottybacks (Pseudochromidae) are a diverse group of mesopredators that feed on cryptic macroinvertebrates and newly recruited fishes. This diet behavior may modify the composition and abundance of cryptobenthic fauna within coral reef ecosystems. Understanding how mesopredators partition their diet and the functional role provided by available prey within reefs can assist in understanding the ecological role these predators contribute to coral reef trophodynamics and the effect of their population changes on the reef ecosystem. To assess the diet of three common Pseudochromis species and two distinct color morphs of P. flavivertex in the Red Sea, I used a combination of i) visual stomach content analysis, ii) stomach DNA metabarcoding (18S, COI), and iii) stable isotope analysis (δ15N, δ13C). I evaluated i) dietary niche breadth, ii) variation in diet composition, iii) degree of dietary specialization, and iv) trophic level. These techniques revealed partitioning in the dietary composition and resource use between P. flavivertex, P. fridmani, and P. olivaceus. Although the two technics used for stomach content analysis did not show differences in the dietary composition within color morphs of P. flavivertex, the isotopic signature showed marked differences in the isotopic niche and resource use between morphs. Resource partitioning appears to be driven by variation in resource availability in the fish habitat and by subtle differences in the ecology of these species. These findings provide evidence of species-specific differences in the trophic ecology of pseudochromids in the Red Sea and demonstrate their important role as predators of cryptic invertebrates and small fish, being key components in energy transfer in coral reef ecosystems by acting as a link between cryptofauna and higher trophic levels. This study highlights the importance of combining several approaches (short-term: visual analysis and DNA metabarcoding; and long-term: isotope analysis) when assessing the feeding habits of coral reef fish, as they provide different and complementary information necessary to delimit their niches and understand the role that small mesopredators play in coral reef ecosystems.