Quantification of key pathways sustaining ecosystem function is critical for underpinning informed decisions on development approvals, zoning and offsets, ecosystem restoration and for meaningful environmental assessments and monitoring. To develop a more quantitative understanding of the importance and variation in food webs and nutrient flows in tropical estuaries, we investigated the spatio-temporal distribution of biomass of fish across 28 mangrove-lined estuaries in tropical Australia. We evaluated the extent to which nekton biomass in tropical estuaries responded to spatial and temporal factors and to trophic identity. Biomass was dominated by two trophic groups, planktivores and macrobenthos feeders. Contributions by other trophic groups, such as detritivores and microbenthos feeders, were more variable. Total biomass and the biomass of all major trophic groups were concentrated in downstream reaches of estuaries. The consistent concentration of biomass downstream, and spatio-temporal differences in the contributions by the different trophic groups, indicates substantial differences in food web structure, differences in the contributions from different sources of nutrition and probably unequal flow of productivity into higher levels of the food web in different parts of the estuary. In turn, this suggests substantial qualitative and quantitative differences in ecosystem-supporting processes in different estuary reaches.