Dottybacks (Pseudochromidae) are a diverse group of mesopredators that feed on cryptic macroinvertebrates and small fishes. Despite their high diversity and endemism in the Red Sea, very little is known regarding the role of dottybacks on coral reef trophodynamics. Understanding how mesopredators partition their diet and the functional role provided by consumed prey within reefs can assist in understanding the ecological role these predators contribute in coral reef trophodynamics. Here, we assessed the diet of Pseudochromis flavivertex, Pseudochromis fridmani, and Pseudochromis olivaceus, and two distinct color morphs of P. flavivertex in the Red Sea using a combination of: i) visual stomach content analysis, ii) stomach DNA metabarcoding (COI), and iii) stable isotope analysis (d15N, d13C). These techniques revealed partitioning in the dietary composition and resource use among the three species.