AbstractThe Sábalo, Prochilodus lineatus (Valenciennes, 1837), is one of 270 fish species in the Rio Paraná system, and it comprises >50% of the fish biomass. Its diet is flocculant benthic biofilm comprised of algae, bacteria and non‐living organic matter: a food resource apparently of little value to other fishes. Digestion and assimilation of key nutrients from its biofilm diet by P. lineatus were described and quantified in an attempt to discover how this species is so successful. The fish begin a feeding period with empty digestive tracts, accumulate food during the feeding period and then void the gut content at the end of the feeding period. Early in a feeding period, sand accumulates in the gizzard‐like pyloric stomach where it serves as both a grinding medium and a sieve. After sufficient sand has been acquired, food particles passed from the pyloric stomach to the intestine are dramatically reduced in size to <20 μ maximum dimension, whereas larger particles including mineral matter and plant fibres are retained. Total ash, hydrolysis‐resistant‐ash and hydrolysis‐resistant‐organic‐matter were tested as reference materials against which to measure assimilation and hydrolysis‐resistant‐organic matter best met the assumptions of the technique. Comparison of the first food ingested to food ingested later in the feeding period shows that grinding of food and selective retention of larger particles results in a three‐fold increase in the assimilation of ash‐free‐dry‐mass (to 56%) and hydrolysis‐labile‐organic‐matter (to 67%), and a six‐fold increase in the assimilation of amino acids (AAs; to 74%). When food quality is assessed in terms of g AA assimilated · kJ−1 energy assimilated, the quality of food ingested by P. lineatus ranges from a maintenance level of 5 to 12 mg AA · kJ−1, a level expected to produce near maximum growth. Thus, the processing of food in the pyloric stomach is integral to the success of P. lineatus in establishing large populations on a diet of flocculent benthic biofilm.