The attachment of microorganisms to electrodes is of great interest for electricity generation in microbial fuel cells (MFC) or other applications in bioelectrochemical systems (BES). In this work, a microcosm of the acidic ecosystem of Río Tinto was built and graphite electrodes were introduced at different points. This allowed the study of electricity generation in the sediment/water interface and the involvement of acidophilic microorganisms as biocatalysts of the anodic and cathodic reactions in a fuel-cell configuration. Current densities and power outputs of up to 3.5 A/m² and 0.3 W/m², respectively, were measured at pH 3. Microbial analyses of the electrode surfaces showed that Acidiphilium spp., which uses organic compounds as electron donors, were the predominant biocatalysts of the anodic reactions, whereas the aerobic iron oxidizers Acidithiobacillus ferrooxidans and Leptospirillum spp. were detected mainly on the cathode surface.