For fishes, recruitment and cohort strength are influenced by survival through early life stages, which, in part, is determined by food availability. As such, knowledge of diet and key prey trophic interactions during early life stages, and how these are influenced by environmental parameters (e.g. river flow), are crucial for understanding population dynamics. We used a combination of traditional microscopy and DNA metabarcoding to investigate the diet of Murray cod (Maccullochella peelii) from the lower River Murray during several stages of early ontogeny (larvae to ~18 months old) and also temporal variability in diet for specific life stages (larvae and ~150 days old) across a seven-year period. Results were generally consistent between methods. Prey contributing greatest (by number of individuals or DNA reads) to larval diet were specific copepods, cladocerans and rotifers, and the shrimp Paratya australiensis. Larvae demonstrated selection (Strauss index) for calanoids Boeckella triarticulata and Calamoecia spp., the cyclopoid Acanthocyclops australis and rotifers of the genus Trichocerca. Diet from 60 days to 18 months was dominated by decapods, with lesser contributions by isopods and fish. With increasing age from 60 days to 18 months, there was a shift in diet from dominance by P. australiensis to the larger decapod Macrobrachium australiense, and increasing contribution of Cherax destructor. The diet of ~150-day-old Murray cod was temporally consistent across 2015–2021 and dominated by decapod shrimp. In contrast, larval diet varied across years under different river flow conditions due to differing contributions of copepods and cladocerans. Our findings have improved understanding of the key food resources of early life stage Murray cod in the lower River Murray and will inform flow management. Flow regimes that promote abundance of preferred prey species may enhance recruitment of Murray cod, and in turn promote population resilience.