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Trophic interactions play a key role in nursery habitats, and by affecting growth and condition of the juveniles, may control the quality of a given estuarine nursery. This study investigated the trophic ecology of flounder (Platichthys flesus) and common sole (Solea solea) juveniles in the Lima estuary nursery. Feeding location, main organic matter sources, and prey of the target species were assessed by carbon (δ13C) and nitrogen (δ15N) stable isotopes, as well as by stomach content analysis. The juveniles, macroinvertebrates, and sediment and water column samples were collected in August 2014 in the lower, middle and upper sections of the Lima estuary. The diet of 0+ flounder relied upon prey from the upper estuary (salinity >5), namely Chironomid larvae and Corophium spp. which showed the role of the upper estuary prey to the 0+ flounder diet and suggest the relative site fidelity of the young juveniles. In contrast, 1+ flounder juveniles had a diverse diet based on bivalves, polychaetes and crustaceans, and a variable stable isotope signature indicating they fed in different areas along the estuary. The 1+ sole juveniles also fed on polychaetes, crustaceans, and bivalves, but the stable isotope values suggested a dependence on the lower estuary (salinity >30) and marine food web sources. Such differential use of food may be understood as a strategic approach to reduce intra- and interspecific competition and thus ensuring the use of Lima estuary as nursery area for these two flatfish species.