Possible use of wine wastes containing ethanol as carbon and energy source for sulphate-reducing bacteria (SRB) growth and activity in the treatment of acid mine drainage (AMD) is studied for the first time. The experiments were performed using anaerobic down-flow packed bed reactors in semi-continuous systems. The performance of two bioreactors fed with wine wastes or ethanol as carbon sources is compared in terms of sulphate reduction, metals removal and neutralization. The results show that efficient neutralization and high sulphate removal (>90%) were attained with the use of wine wastes as substrate allowing the production of effluents with concentrations below the required local legislation for irrigation waters. This is only possible provided that the AMD and wine wastes are contacted with calcite tailing, a waste material that neutralizes and provides buffer capacity to the medium. The removal of metals using wine wastes as carbon source was 61-91% for Fe and 97% for both Zn and Cu. The lower removal of iron, when wine waste is used instead of ethanol, may be due to the presence of iron-chelating compounds in the waste, which prevent the formation of iron sulphide, and partial unavailability of sulphide because of re-oxidation to elemental sulphur. However, that did not affect significantly the quality of the effluent for irrigation. This work demonstrates that wine wastes are a potential alternative to traditional SRB substrates. This finding has direct implication to sustainable operation of SRB bioreactors for AMD treatment.