An integrated assessment of sediment quality in the Guadiamar River after a mining spill was conducted. The concentration of different metals and other conventional parameters were measured in sediments located along the river. Four sediment toxicity tests (Hyalella azteca 28-day survival and growth test; Chironomus riparius 10-day survival and growth test; Hexagenia spp. 21-day survival and growth test; and Tubifex tubifex 28-day reproduction and survival test) were carried out to determine the effects associated with the accidental spill. The geochemical fractions of 6 metals (Fe, Mn, Zn, Cu, Pb, and Cd) were determined to establish the bioavailability of the metals. The relationship found in the concentrations of metals associated with the mobile fractions of the sediments in the sites studied is similar to the toxic mud from tailing pond and confirms that the toxic effects are associated with the metals Zn and Cd originating from the spill.