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Microbial communities of iron-rich water in the Pyhäsalmi mine, Finland, were investigated with high-throughput amplicon sequencing and qPCR targeting bacteria, archaea, and fungi. In addition, the abundance of Leptospirillum and Acidithiobacillus was assessed with genus-specific qPCR assays, and enrichment cultures targeting aerobic ferrous iron oxidizers and ferric iron reducers were established. The acidic (pH 1.4-2.3) mine water collected from 240 m, 500 m, and 600 m depth from within the mine had a high microbial diversity consisting of 63-114 bacterial, 10-13 archaeal, and 104-117 fungal genera. The most abundant microorganisms in the mine water were typical acid mine drainage (AMD) taxa, such as acidophilic, iron-oxidizing Leptospirillum, Acidiphilum, Acidithiobacillus, Ferrovum, and Thermoplasma. The fungi belonged mostly to the phylum Ascomycetes, although a great part of the fungal sequences remained unclassified. The number of archaeal 16S rRNA genes in the mine water was between 0.3 and 1.2 × 10 7 copies mL −1 in the samples from 500 m and 600 m, but only 3.9 × 10 3 at 240 m and archaea were in general not enriched in cultures. The number of fungal 5.8S rRNA genes was high only in the mine water from 500 m and 600 m, where 0.2-3.4 × 10 4 spore equivalents mL −1 were detected. A high number of Leptospirillum 16S rRNA genes, 0.6-1.6 × 10 10 copies mL −1 , were detected at 500 m and 600 m depth and in cultures containing ferrous iron, showing the importance of iron oxidizers in this environment. The abundance of bacteria in general was between 10 3 and 10 6 16S rRNA gene copies mL −1 . Our results showed a high microbial diversity in the acid-and iron-impacted waters of the Pyhäsalmi mine, where Leptospirillum bacteria were especially prominent. These iron oxidizers are also the main nitrogen-fixing microorganisms in this ecosystem.