Total contents of 36 potentially toxic elements are summarized for agricultural topsoil (n = 12; soil depth = 0–20 cm), subsoil (n = 12; soil depth = 20–40 cm), and representative rock samples collected from a Mediterranean site (Megara Plain, Greece). The five-stage sequential extraction procedure for the geochemical partitioning of cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), and nickel (Ni), proposed by Tessier, was applied to topsoil and subsoil collected from the study area. Soil Cd was highly associated with exchangeable fraction, illustrating high bioavailability of this element. The order of mobility of the elements was as follows: Cd > Cu > Co > Zn > Ni > Cr > Mn. Results from sequential extraction experiments illustrated that the bioavailability of Cu, Co, and Zn is moderate, while Ni, Cr, and Mn presented low bioavailability, indicating that these elements could pose a limited threat to the quality of crops. Cadmium is the chief contamination controlling factor posing moderate potential ecological risk. The contamination sources of the examined elements are discussed.