The raw data consisted of demultiplexed fastq files pairs (R1.fastq and R2.fastq) per sample accessible on the NCBI Sequences Read Archive (SRA) under the BioProject accession numbers PRJNA1187555 for experiments E1 and E3 and PRJNA1187576 for E2 and E4. This dataset is associated with the following publication: Valentin, V., S. Rivera, E. Acs, S. Almeida, K. Andree, L. Apothéloz-Perret-Gentil, B. Bailet, A. Baričević, K. Beentjes, J. Bettig, A. Bouchez, C. Camilla, C. Chardon, M. Duleba, T. Elersek, C. Genthon, M. Jablonska, L. Jacas, M. Kahlert, M. Kelly, J. Macher, F. Mauri, M. Moletta-Denat, A. Mortágua, J. Pawlowski, J. Pérez-Burillo, M. Pfannkuchen, E. Pilgrim, P. Panayiota, F. Rimet, K. Stanic, K. Tapolczai, S. Theroux, R. Trobajo, B. Van der Hoorn, M. Vasquez, M. Vidal, D. Wanless, J. Warren, J. Zimmermann, and B. Paix. Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring. Metabarcoding and Metagenomics. Pensoft Publishers, Sofia, BULGARIA, e133264, (2025).