These data consist of raw 16S rRNA gene sequences for the bacterial communities in three upland Welsh river sites under different treatments. A mapping file with metadata for each sample is provided and a operational taxonomic unit (OTU) table. These sites were situated in three streams from the Llyn Brianne Stream Observatory, Powys, Wales, UK (52°08' N, 3°45' W). The catchments cover approximately 300 square kilometres of upland Wales in the upper Afon Tywi. These first to third order experimental streams rise in either rough, sheep-grazed moorland (named as L6 and L7) or plantations of Sitka spruce Picea sitchensis with lodgepole pine Pinus contorta (named as L3). Some reductions of forest cover have occurred in L3 with normal logging operations. A 24-hour experiment was conducted at the Diversity in Upland Rivers for Ecosystem Service Sustainability (DURESS) cascading flumes at these streams during September 2014. Each flume consisted of 3 channels, each assigned a different treatment: control, sugar addition and peat addition. Sugar (sucrose) and peat were added to channels to represent a simple and complex form of dissolved organic carbon (DOC) respectively. Five biofilm samples were collected from random locations in each experimental channel. Samples were taken at 0.5, 3, 15 and 24 hours after the start of the experiment. Epilithon were taken from unglazed ceramic tiles that had been colonised by epilithon in the river. After amplification, the 16S rRNA fragments were sequenced on the Illumina MiSeq next generation sequencing platform. The main goal of this survey was to characterise bacterial diversity, the chemical and biological consequences of elevated DOC inputs, and to investigate the role of bacterial organisms in controlling organic carbon flux. Prof Andy Weightman and Dr Isabelle Durance were responsible for organising the experiments. Sampling was carried out by Dr. Isa-Rita Russo and a team of Post Doctoral Research Assistants (PDRA's)/students. The work was carried out under the Diversity in Upland Rivers for Ecosystem Service Sustainability (DURESS) project (Grant reference NERC NE/J014818/1). DURESS was a project funded by the Natural Environment Research Council (NERC) Biodiversity and Ecosystem Service Sustainability (BESS) programme. Full details about this dataset can be found at https://doi.org/10.5285/df829b9f-c4c5-4e53-9217-c9c1e5bd078d