Fatty acids are the main components of lipids and are necessary for the production and permeability of cell membranes, playing an essential role in the physiological responses of organisms. The biochemical composition of zooplankton might be used as bio-indicators of the trophic status of aquatic ecosystems. Aiming to fill the gap of knowledge in tropical estuaries, the main aim of this study was to test if the fatty acid profiles can reveal spatial and temporal shifts in the diet of copepods and therefore can be used as indicators of the trophic status of estuarine systems. We investigated the fatty acids composition of copepod species and their possible food sources along the salinity gradient of two tropical estuaries (Paraíba do Norte and Mamanguape estuaries, northeastern Brazil), during the rainy and dry seasons. We found clear seasonal differences regarding fatty acids composition and concentration in copepods, with maximal concentrations and diversity of total fatty acids during the rainy season. The copepods species were mainly carnivorous in the dry season and omnivorous in the rainy season and, in both estuaries, the diet of most copepods was dependent on food availability. The fatty acid profiles suggest that, in general, feeding patterns of zooplankton change spatially and temporally, reflecting the shifts in their food sources abundance (i.e., dominance among diatoms and flagellates, terrestrial detritus and small animals). We observed a residual proportion of terrestrial detritus and green algae in the diets and these items were only present in the dry season. Furthermore, the food sources in the Paraiba do Norte estuary, a system with high levels of anthropogenic disturbance, showed lower feeding quality, with lower essential fatty acids concentrations. Our study showed that fatty acid profiles can be used as ecological indicator to assess seasonal and spatial shifts in the trophic ecology of copepods in tropical estuaries and to distinguish systems with different levels of human impact in a fast and accurate way.