The microbial leaching of manganiferous minerals originating from Greece and Italy has been the subject of EU research for about 3 years, in order to develop an economical process of biotreatment. A heterotrophic mixed culture, selected from natural water was found to be capable of reducing the MnO2 in microaerobic conditions. The first culture medium, formulated in a preliminary way, was employed in the selection and cultivation of the microbial strains in the first steps of this study. For this reason an optimization study of the culture media was carried out by means of fractional and full factorial experiments in order to establish the correct medium composition for the biotreatment. The bioleaching tests were carried out in shake flasks using a low grade Italian mineral. The factors analysed were the concentrations of minerals, molasses, NH4NO3, KH2PO4, MgSO4, yeast extract and NaHCO3. The manganese extraction yield was the response of the process. A reduction of the salt concentrations present in the media was found to be possible with a significant reduction in cost. The experimental results show the potential application of the mixed culture for the bioleaching of manganiferous minerals not treatable by means of conventional processes.