The Pyhäsalmi mine, central Finland, has operated as a deep metal mine since 1967. It currently reaches a depth of almost 1500 m, making it the deepest mining operation in Europe. Around 900,000 m3 of metal-rich, extremely acidic water are pumped out of the mine each year. The near constant air temperature of ∼24 °C together with exposure of sulfidic rock surfaces to air and water, have created an environment that is highly suitable for colonization by acidophilic mineral-oxidizing microorganisms. Using a combined cultivation-dependent and molecular approach, indigenous bacteria in waters at two depths within the mine, and of an acid streamer sample were identified and isolated. Iron-oxidizing chemolithotrophs (Acidithiobacillus and Leptospirillum spp., and “Ferrovum myxofaciens” were the most abundant bacteria in mine water samples, whereas the acid streamer community contained a greater proportion of heterotrophic acidophiles (Ferrimicrobium acidiphilum and a gammaproteobacterium related to Metallibacterium scheffleri). The most abundant isolates obtained from both water and streamer samples were all strains of Acidithiobacillus Group IV, a proposed separate species of iron-oxidizing acidithiobacilli that has not yet been classified as such. Archaea were also detected in water and streamer samples using molecular methods, but most were not identified and no isolates were obtained.