Ash-free dry mass (AFDM) of three distinct size fractions of organic matter (>1cm, >1mm and > 0.025mm) collected in quantitative samples of the benthic substrate determined from up to 10 replicate samples at three chalkstream sites in the Wessex chalk area (Nine Mile River, River Till and River Wylye) on each sampling occasion. Data were collected on seven occasions on the Wylye and Nine Mile River and on five occasions on the Till, between October 2012 and October 2013. Data were collected to construct quantified food webs detailing the flux of mass and nutrients between nodes of the food web. This dataset was created as part of work package 3.2 of the Wessex Biodiversity & Ecosystem Service Sustainability (BESS) project.