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Aljustrel mining area (South Portugal) is a part of the Iberian Pyrite Belt and encloses six sulfide mineral masses. This mine is classified of high environmental risk due to the large tailings' volume and acid mine drainage (AMD)-affected waters generated by sulfides' oxidation. The use of biological indicators (e.g., diatoms) revealed to be an important tool to address the degree of AMD contamination in waters. Multivariate analysis has been used as a relevant approach for the characterization of AMD processes. Cluster analysis was used to integrate the significant amount and diversity of variables (physicochemical and biological), discriminating the different types of waters, characterized by the high complexity occurring in this region. The distinction of two main marked phenomena was achieved: (1) the circumneutral-Na-Cl water type (sites DA, PF, BX, BF, RO, CB), expressing the geological contributions of the Cenozoic sediments of Sado river basin, with high diatom diversity (predominating brackish diatoms as Entomoneis alata); and (2) the acid-metal-sulfated water type (sites BM, JU, RJ, AA, MR, BE, PC, AF), reflecting both the AMD contamination and the dissolution of minerals (e.g., silicates) from the hosting rocks, potentiated by the extremely low pH. This last group of sites showed lower diatom diversity but with typical diatoms from acid- and metal-contaminated waters (e.g., Pinnularia aljustrelica). In addition to these two water types, this hierarchical classification method also allowed to distinguish individual cases in subclusters, for example, treated dams (DC, DD), with alkaline substances (lime/limestone), that changed the physicochemical dynamics of the contaminated waters.