Intensive mining has taken place in the Iberian Pyrite Belt since 3000 B.C. generating Acid Mine Drainage (AMD) and releasing high amounts of SO4, acidity, metals and metalloids into surface water. Concentrations of elements in AMD-impacted waters are regulated by the precipitation of Fe-rich materials and particulate matter can influence the mobility and the bioavailability of metals. In this paper a study on the dissolved As species concentration along a polluted stream has been performed. Two sampling campaigns were conducted during the dry and the rainy seasons. Concentrations of dissolved elements are higher during the dry season and increase progressively along the water course in both seasons. Concentrations up to 80 μg L−1 of As3+ and 5 mg L−1 of As5+ were determined. The concentration of As species increases and the As3+/As5+ ratio decreases downstream. The Fe2+/Fe3+ and As3+/As5+ ratios show the same pattern with respect to pH for all the examined samples, except those taken from Au cyanidation wastes. The particulate phase is mainly composed of Fe, As and Pb, with As being associated with the Fe minerals while Pb seems to be associated with the clay colloids.