Chlorophyll-a content and ash-free dry mass of quantitative samples of the biofilm on benthic substrate from three chalkstream sites in the Wessex chalk area (Nine Mile River, River Till and River Wylye). Chlorophyll a content and biomass were determined from up to 10 replicate samples at each site on each sampling occasion. The data are presented as means with standard deviation. 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. Full details about this dataset can be found at https://doi.org/10.5285/64a8cef6-2d5b-452b-8f60-0cfa85ae51bc
