Few studies have been made on planktonic food webs of temperate ecosystems, especially those from the Southern Atlantic Ocean, using molecular biomarkers. The fatty acid compositions of suspended particulate matter (SPM), microplankton and mesozooplankton were studied during summer and winter at a sewage-impacted and a control site in the Bahıa Blanca Estuary (Argentina). The aim was to identify trophic relationships on a spatial and seasonal scale and to detect allochthonous inputs to the food web. Fatty acid trends were consistent with the seasonal succession of the plankton community structure supporting our underlying hypothesis that regional seasonality is mostly responsible for changes in fatty acid composition. Sewage had no clear impact on the fatty acids and may not be a significant source of SPM in the estuary. However, at the sewage site the composition of the SPM was more related to terrestrially derived compounds, diatoms and bacteria, and mesozooplankton fatty acids suggested grazing on terrestrial components and on diatoms over flagellates. Saltmarshes likely have a crucial role as the main contributors to the organic fraction of SPM followed by plankton. The seasonal fatty acid pattern of the mesozooplankton indicated different feeding strategies suggesting an active feeding mode during summer and a more terrestrially associated diet in winter. The fatty acid trophic marker approach provided relevant information to clarify planktonic trophic interactions and to trace the origin of organic matter in this highly dynamic temperate coastal system.