Studies on food web structure in estuaries based on stable isotopes have usually not taken spatiotemporal variations in the isotopic variability of food web components into account. We investigated temporal and between-habitat variations in the isotopic composition ( 13 C/ 12 C and 15 N/ 14 N) of basal food sources -i.e. primary producers, particulate organic matter (POM) in the sediment or in suspension, and biofilm -and consumers and explored their implications for food web structure and dynamics. Samples of 9 basal sources (N = 224) and 6 representative invertebrates and fish (N = 303) were collected monthly over 1 yr in a marsh creek and a mudflat of the Patos Lagoon, a large southwestern Atlantic estuary. The relative contributions of food sources to consumers were estimated by mixing models. Overall, δ 13 C values of food sources and consumers were higher (p < 0.05) in the mudflat, whereas δ 15 N values were enriched (p < 0.05) in the marsh creek for most primary producers and consumers. C and N isotope values varied on both monthly and seasonal scales, with several food sources and consumers exhibiting higher values (p < 0.05) during the spring and summer. The primary food sources were POM in the sediment, C 4 plants (e.g. widgeon grass), and, to a lesser extent, suspended POM. Biofilm and macroalgae were also food sources for consumers in the marsh creek and mudflat, respectively, especially during warmer seasons. Our findings corroborate the hypothesis that the Patos lagoon food web is spatially structured and supported by local basal sources and detritus of different origins, which can be seasonally important for consumers in each particular habitat.KEY WORDS: Estuarine food web · Salt marsh detritus · Mixing model · SIAR · Widgeon grass · Biofilm · Fish · Decapod crustaceans · Temporal variability Resale or republication not permitted without written consent of the publisher