Acidophiles play a vital catalytic role in the formation of acid mine drainage (AMD). In this study, acidophilic communities in a stream, two lakes and five ponds in Nanshan Mine, Anhui Province, China were comparatively investigated. Among the three types of AMDs, the stream which originated from a rock wall had the strongest acidity (pH 2.30–2.42), while the ponds (pH 2.35–2.60) formed in mine dump and the two large-scale AMD lakes (pH 2.66–3.05) were less acidic. The chemolithotrophic iron-oxidizer Leptospirillum dominated at the stream starting point with the lowest pH and highest Fe content. However, with streamflow, pH increased and Fe concentration decreased dramatically, and the dominance of Leptospirillum was replaced by the less acid-resistant iron-oxidizer Ferrovum. In contrast to the stream, prokaryotic communities in the ponds were mostly dominated by chemoheterotrophic Acidiphilium because of the abundant organic matter from surrounding mine waste. Both chemolithotrophic Ferrovum and chemoheterotrophic Acidiphilium were abundant in the AMD lakes, which may form a symbiotic relationship around the carbon cycle. Statistical analyses indicated that pH was the predominant environmental factor influencing the prokaryotic communities. As for the eukaryotic communities, the microeukaryotes in the stream were mostly uncultured groups. Both photoautotrophic algae Chrysonebula and chemoheterotrophic fungi were abundant in the ponds, while the two AMD lakes were only dominated by photoautotrophic algae Coccomyxa and Ochromonas.