The mixing pattern along a summer salinity gradient in the estuary Odense Fjord was evaluated using nutrient concentrations as well as 13C and 15N isotope signatures of suspended and sediment organic matter, immobile macrophytes (Fucus vesiculosus and Ruppia maritima), and benthic fauna (Mya arenaria, Hediste (Nereis) diversicolor, and Arenicola marina). Trophic discrimination (Δ13C and Δ15N) of the infaunal consumers (suspension feeders and detritivores) was assessed from the obtained mixing patterns along the estuarine gradient. Correspondence between salinity, DIC, and DIN in Odense Fjord implies conservative mixing as also evident from linear relationships between salinity and δ13C and δ15N signatures of most living organic pools. Isotope signatures of suspended organic matter (i.e., diatoms) indicate that the river to marine DIC and DIN end-members have daily/weekly δ13C and δ15N averages during summer from − 10 to 0‰ and 10–12 to 0–5‰, respectively. Stable isotope signatures of long-lived macrophytes stationary at specific locations in Odense Fjord showed δ13C levels that were about 7‰ higher than for suspended particles and 3–4‰ higher than for sediment organic matter, while no such difference was evident for δ15N. The food of invertebrate consumers (M. arenaria, H. diversicolor, and A. marina) determined from the estuarine δ13C and δ15N patterns provided the first ever reported trophic discrimination of these animals. Thus, Δ13C was 1.9, 1.6, and 1.3‰ and Δ15N was 4.4, 5.0, and 3.5‰ for the three species, respectively. Accordingly, benthic suspension and deposit feeders in Odense Fjord are largely supported by a diet consisting of benthic and pelagic microalgae, however, with a possible slight shift in diet proportions or to other food sources in the lower reaches of the estuarine gradient.