A 1.2 km long effluent from La Zarza-Perrunal mine (Iberian Pyritic Belt, IPB) was characterized and compared with Río Tinto. In La Zarza effluent microbial oxidation of ferrous iron is responsible for the drastic increase in ferric iron, from a ratio of Fe(III)/Fetotal of 0.11 at the origin, up to 0.99 downstream. Prokaryotic and eukaryotic diversity throughout the effluent were determined. Bacteria related to the sulfur cycle as well as iron-reducing bacteria were mainly detected near the anoxic origin. Iron-oxidizing microorganisms increased along the course of the effluent following an increase in the oxygen content in the water column. Eukaryotic diversity varied drastically along the effluent. Rio Tinto (92 km length) is a natural extreme acidic environment with a rather constant acidic pH (mean pH value 2.3) and a high concentration of heavy metals. The Tinto ecosystem is under the control of iron . The geomicrobiological comparisons of both habitats were performed to unravel some basic questions of biohydrometallurgical interest.