1) The food web of the central Amazon basin displays one of the largest discrepancies in food source utilisation versus availability for consumers. While C4 macrophytes dominate the primary producing biomass in floodplains, the food web is dominated by the use of C3 carbon sources. Amazon fish species have wide-ranging diets and show feeding flexibility in response to spatial and temporal patterns in food source availability. Fish are therefore expected to use a range of available resources. Fatty acids and stable carbon and nitrogen isotopic compositions were studied to investigate their trophic behaviour and provide a better assessment of food source utilisation by nine fish species from two Amazon floodplains. 2) Although nitrogen stable isotopes positioned detritivores at the base of the food chain, their large proportions of polyunsaturated fatty acids (FAs) suggest selection of a high-quality food source, such as microalgae. Omnivores and insectivores displayed a wide range of food source utilisation, indicating opportunist feeding behaviour. Piscivores displayed the most 15N-enriched signature (up to 10.6‰), concomitant with their predator position in the studied food web. 3) C3 carbon was the dominant source for most species, which is a finding consistent with current classical knowledge. However, consumption of C4 macrophytes was detected for the herbivore Schizodon fasciatus, in line with the spatial availability of this food source in the floodplain system. Large amounts of polyunsaturated and essential FAs in these C4 macrophytes indicate their high nutritive value. Our results suggest that this food source is rather neglected by fish due to low digestibility, with the exception of the herbivore that may possess modification of its digestive system that allows the use of C4 plants