Obligate autotrophic and acidophilic characteristics of iron oxidizing bacteria were exploited to prevent or attenuate the generation of AMD. Inhibition of iron oxidizing bacteria growth was performed by variation of growth medium by increasing pyrite concentration (substrate inhibition), by addition of limestone (inhibition by pH increase) and olive pomace (inhibition by organic compounds). Preliminary pyrite bioleaching tests showed the ability of the available inoculum to oxidize the mineral. Inhibition tests of batch growth were performed according to full factorial design with three factors (pyrite, limestone and olive pomace). Experimental data denoted a strong inhibition in all the operating conditions, except the case when larger pyrite concentrations were used. In particular, limestone is a strong inhibitor and inhibition by olive pomace may be related to the antioxidant properties of polyphenols. Interactions among operating conditions were specifically assessed by analysis of variance.