Introduction: DNA metabarcoding is currently under intense scrutiny for its future implementation in biodiversity monitoring and ecological assessment, particularly within the EU Water Framework Directive (WFD). However, before metabarcoding can be deployed for WFD reporting, challenges such as the incompleteness of reference databases need to be addressed. In this context, this study presents the results of a national barcoding project (FRESHBAR) focusing on benthic freshwater diatoms of Sweden, a key organism group regarding both ecology and environmental assessment. One major goal of the project was the publication and vouchering of all data, material, and results. Methods: A total of 312 diatom cultures were established, with a focus on oligotrophic and acid habitats. The cultures were sequenced for two barcodes (rbcL and 18SV4) and identified using light microscopy; selected strains were also studied by scanning electron microscopy. All data, including sampling metadata, barcode sequences, images, and voucher material, were published following the FAIR principles. Some cultures were archived in diatom culture collections. Results: Almost all strains were successfully sequenced and identified as 51 taxa across 17 genera, with the highest diversity in the genera Eunotia and Fragilaria. An attempt was also made to identify all taxa at the species level. Approximately half of the taxa were new to Diat.barcode, a curated diatom reference database. Conclusion: FRESHBAR represents the first large-scale effort to generate barcode reference sequences for Nordic benthic freshwater diatoms. As all data are publicly available, the added sequences and morphological information will help identify all taxa properly to the species level, unravel taxon relationships within diatoms, and improve reference databases for environmental monitoring and research.