The maintenance of species diversity in a given environment is strongly linked to resource partitioning. Littoral macrophyte zones are heterogeneous environments with high microcrustacean diversity, where zooplankton have dietary access to seston as well as organisms growing on macrophytic surfaces (epiphyton). We conducted a field study in a macrophyte-rich backwater of the river Allier to examine how seston and epiphyton were used as potential food sources by four dominant cladoceran species. Fatty acids were analyzed in these two food sources to assess how their differential uptake affects the trophic trajectory of essential compounds from these resources to cladocerans. Our results showed resource partitioning among the four cladocerans studied; while Eurycercus fed mostly on epiphyton, Daphnia mostly consumed phytoplankton, and Ceriodaphnia and Simocephalus were able to forage on sestonic and epiphytic resources. Based on their polyunsaturated fatty acid (PUFA) content, it was evident that epiphyton was of higher food quality than seston in this macrophyte-rich backwater system. Variability of PUFA compositions of seston and epiphyton, and diversity of foraging strategies of cladoceran species, which represent the major link between microorganisms and consumers at higher trophic levels, affect dietary energy pathways and point to a variable PUFA transfer efficiency in backwater food webs.