The present work deals with a bioremediation study of a heavy-metal polluted harbour sediment, obtained from the Italian Adriatic Coast. Bioleaching of the sediment sample was performed with a mixed culture of acidophilic, chemi-autotrophic Fe/S oxidising bacteria. The effect of an anaerobic biostimulation pre-treatment on the extent of Cd, Cu, Zn, Ni, Pb, Hg, As, Cr extraction by bioleaching was evaluated. The biostimulation pre-treatment was intended to stimulate autochthonous sulfate reducing strains, to enhance the sulfide fraction in the sediment, to favour subsequent activity of reduced-sulfur-oxidizing bacteria in the subsequent bioaugmentation (bioleaching). The effect of the duration of anaerobic pre-treatment (21 and 30 days) in the presence and absence of 1% glucose was tested. The results obtained showed that the activity of the reduced-sulfur-oxidising strains was significantly enhanced after an anaerobic pre-treatment of the sediments and showed real promise for the application of bioleaching for metal polluted sediments.