Differential utilization of isomers pyrite and marcasite by Acidianus manzaensis were comparatively studied, besides the iron and sulfur speciation transformation of the minerals was also investigated based on synchrotron radiation X-ray absorption near edge structure (XANES) spectroscopy. The results showed that the biooxidation of pyrite was faster than marcasite. The bioleached surface of both pyrite and marcasite are serious corroded, and the Fe (III)-containing species as well as jarosite were gradually produced, and more elemental sulfur species were formed on the marcasite surface than that for pyrite. It demonstrates the mineral structure does affect the biooxidation of pyrite and marcasite, and the more precipitated elemental sulfur might be one of the reasons leading to lower oxidation rate of marcasite by A. manzaensis.