The mobility and bioavailability of As and Sb in relation to soil-biota transfer were evaluated at a former Sb mining and smelting site (Glendinning, SW Scotland, UK). The study specifically assessed the accumulation of As and Sb in different environmental components (soil, plants and earthworms) across mining area to estimate risk factors for the biota. Total concentrations and fractions of As and Sb in soils were determined. The latter using both a single solute and a non-specific stepwise sequential extraction (CISED) method. Mineralogical information was gathered from XRD and SEM analysis used to identify element distribution patterns. Pseudo-total (aqua-regia) levels of As and Sb in the soils varied between 50–17,400 mg kg− 1 and 10–1,200 mg kg− 1, respectively. Both elements are predominantly associated with Fe (or Al) oxides/hydroxides, by adsorption around silicate grains, representing a potentially bioavailable fraction. Antimony was also associated with sulphide phases. The highest values of As and Sb in biota were recorded in the earthworms (960 mg kg−1 and 27 mg kg− 1, respectively). Bioconcentration factors for both elements were below 1 and the highest for earthworms. Total and leached As levels in soils and biota were positively and significantly correlated, but only for Sb in earthworms and grass. Bioavailability of As in the biota, was shown to be limited by pH. In spite of the considerably high As and Sb contents of the soil the plant contamination remained comparably low, but still exceeded background values.