The Guadalquivir LTER is a Long Term Ecological Research program monitoring monthly since 1997 the biodiversity, population densities, environmental variables, community and trophic structure of the Guadalquivir river estuary. This last section of 50 km of “the big river” is a highly dynamic ecosystem which provides relevant ecosystem services to society, especially as a critical nursery ground for many fish and crustacean species. The estuary presents a high productivity, a marked seasonal cycle defined by temperature while species are clearly spatially distributed along the salinity gradient. More than 350 species are structured in a highly-connected food web fueled by phytoplankton and a heterotrophic microbial community. Food resources are mainly channeled through copepods and mysids. Being aware of the importance of the nutritional value at the base of the food web for an efficient transmission to consumers, a novel quantitative fatty acid–based procedure allowed to quantify both phytoplankton functional groups and the nutritional quality of suspended particulate organic matter (SPOM). The structure of the estuarine phytoplankton community was mostly influenced by salinity; secondly influenced by water temperature, irradiance, and river flow, and unaffected by nutrients. Twelve phytoplankton groups were quantitatively distinguished by iterative matrix factor analysis of seston fatty acid signatures in this turbid estuary. Diatoms dominated about half of the phytoplankton community spatiotemporally. Cyanobacteria and Chlorophytes were more abundant in the upper reaches of the estuary and were little affected by seasonality. Euglenophytes were also more abundant at lower salinity, increasing their presence in autumn–winter. Coccolithophores and Dinophytes contributed more to phytoplankton community at higher salinity and remained little affected by seasonality. Salinity was the main driver of SPOM nutritional quality, with salinities within 20-35 ppt producing SPOM 2.2-fold higher in nutritional value than salinities within 0-5 ppt. SPOM in summer showed a 1.5-fold higher nutritional value than SPOM in winter. Extracting the lipid fraction of SPOM led to estimations of microbial community representing from 20.9% of particulate organic carbon (POC) in winter to 44.5% in summer, and from 28% at salinities of 0-20 ppt to 41% in the 20-35 salinity range. Bacteria-specific fatty acids indicated that phytoplankton maximally contributed to microbial community slightly more than 60% only in summer and at salinities higher than 10 ppt in all seasons. Basal resources in the estuary are mainly channeled to upper trophic levels by mysids, which actively graze on phytoplankton and heterotrophic microorganisms. Mysids are the most abundant and key species in the estuarine food web and represent the main prey for early life stages of many marine fish and crustaceans, especially for the European anchovy Engraulis encrasicholus, a species of great ecological and economic importance in the Gulf of Cadiz marine ecosystem. Long term series analysis of estuarine environmental variables, anchovy juvenile biomass in the estuary and fishery stock biomass show how reduction in freshwater discharges due to damming of the Guadalquivir river (since 1930) has led to decreases in anchovy juveniles in the estuary. It seems likely that trophic cascading up effects associated to altered microbial communities might have influenced on mysid populations decreasing. By considering, the socio-economic-ecological impacts of different management strategies, we show that terrestrial and marine resources cannot be continued to be managed separately without negative consequences for human activities, and propose an Integrated Ecosystem Management for the region.