Soil profiles located in the mining district of Almadén were investigated for total Hg, organic Hg fraction and Hg distribution by selective sequential extraction. A four-step sequential extraction method (labile Hg species, humic and fulvic complexes, elemental Hg and bound to crystalline oxides and Hg sulfide and refractory species) was performed. Total Hg concentrations ranged from 13 to 64 mg per kg dry mass. A clear relationship between the depth and Hg content was found since Hg concentration decreases downwards, which is indicative of anthropogenic contamination via deposition processes from nearby mine waste. Significant organic Hg concentrations were found in all the tested soil profiles ranging from 79 to 287 μg kg−1 (dry weight). It seems that organic Hg was strongly influenced by elemental Hg (r = 0.79) and to a lesser extent by the organic carbon content (r = 0.57). The fractionation revealed that Hg exists mainly as cinnabar in the studied soils, which is one of the least available and mobile Hg species, and as elemental Hg as well. The most mobile Hg fractions only accounted for 3.2 to 7.7% of the total Hg content, with the main contribution being the humic and fulvic complexes fraction. The elemental Hg fraction increased with depth indicating a migration to deeper soil layers. In contrast, the surface layers showed an enrichment in the fraction bound to sulfide, which means that Hg is mostly deposited as cinnabar particles from non-processed ore in this area.