Mineral bio-oxidation improves the extraction of valuable metals and also decreases the impact caused by mining waste; however, the interactions between the micro-organisms so involved are little known. Double-layer solid culture media techniques and amplified ribosomal DNA restriction enzyme analysis (Ardrea), using Eco72I, Eco24I, XcmI and BsaAI enzymes, were used for characterising four micro-organisms isolated from gold mines located in Marmato, Colombia. This work was aimed at better understanding of native acidophilic micro-organisms’ microbial interactions in mix ed cultures . Iron and sulphur oxidising isolates revealed similar restriction patterns to those previously reported for Acidithiobacillus ferrooxidans; however, one of them exhibited different colony morphology compared to previously reported morphology. The iron non-oxidising isolate presented a restriction pattern agree