<p>The discharge of acid mine drainage (AMD) from abandoned mining sites remains a major cause of water quality degradation, particularly in mine-affected regions such as the Iberian Pyrite Belt. The effectiveness of a full-scale passive treatment plant employing the Dispersed Alkaline Substrate (DAS) technology was assessed through a combined chemical and biological monitoring program at the Esperanza Mine (SW Spain). Water samples were collected at an abandoned mine shaft, at the output of the treatment plant as well as at the discharge point into the receiving river. Physicochemical analyses revealed the DAS system successfully neutralized acidity, reduced electrical conductivity, and substantially lowered the concentrations of dissolved and bioavailable metals, including Fe, Zn, and Cu. Acute toxicity tests conducted with a range species (<i>Vibrio fischeri</i>, <i>Raphidocelis subcapitata</i>, <i>Lemna minor</i>, <i>Artemia</i> sp., <i>Corbicula fluminea</i>, and <i>Gambusia holbrooki</i>) showed a clear improvement in organism survival and physiological responses post-treatment. In addition, passive samplers settled in test samples to determine metal availability showed good performance for most elements (especially for Cu), but some limitation with the very acidic pH test samples. Multivariate statistical analyses distinguished acidity and metal load as the primary drivers of toxicity, both effectively mitigated after the DAS treatment. Although treated waters approached the quality of natural reference sites, residual sublethal effects suggest the need for continued ecological monitoring. These findings demonstrate the potential of DAS-based passive treatments to restore chemical and ecological conditions in AMD-impacted systems and contribute to broader environmental objectives, including compliance with the European Water Framework Directive.</p>