The function of the microbiomes in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of their members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), high resolution taxonomic classification is crucial to understand the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on full-length 16S rRNA gene amplicon sequence variants (FL-ASVs) derived from activated sludge and anaerobic digester systems in Denmark. The new database proposes unique provisional names for all unclassified microorganisms down to species level, providing a new and much-needed tool for microbiome research. The MiDAS 3 database was used to analyze the microbiome in 20 Danish wastewater treatment plants with nutrient removal, sampled over 13 years. The 50 most abundant species belonged to 42 genera, including 14 genera with provisional ‘midas’ name. Of those, 20 have no known function in the system, which highlights the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The new MiDAS 3 database also forms the backbone of the MiDAS Field Guide – an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1800) and species (>4200) found in activated sludge and anaerobic digesters in Denmark, but is also relevant to wastewater systems across the world. The identity of the microbes is linked to functional information, where available, and the website provides the possibility to BLAST new sequences against the MiDAS 3 database. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository, facilitating understanding of wastewater treatment ecosystem function.