Ubiquitous presence of microbes in aquatic systems and their inherent ability of biomineralization make them extremely important agents in the geochemical cycling of inorganic elements. However, the detailed mechanisms of environmental biomineralization (e.g., the actual reaction rates, the temporal and spatial dynamics of these processes) are largely unknown, because there are few adequate analytical techniques to observe the biogenic oxidation/reduction reactions in situ. Here, we report a novel technical approach to characterize specific biominerals associated with a target microbe on high spatial resolution. The technique was developed by combining directly in situ phylogenetic analysis, fluorescence in situ hybridization (FISH), with a synchrotron microprobe method, micro X-ray absorption fine structure spectroscopy (μ-XAFS), and was applied to iron mineral deposition by iron(II)-oxidizing bacteria (IOB) in environmental samples. In situ visualization of microbes revealed that in natural iron mats, Betaproteobacteria dominated by IOB were dominantly localized within 10 μm of the surface. Furthermore, in situ chemical speciation by the synchrotron microprobe suggested that the Fe local structure at the IOB accumulating parts was dominantly composed of short-ordered Fe-O(6) linkage, which is not observed in bulk iron mat samples. The present study indicates that coupled XAFS-FISH could be a potential technique to provide direct information on specific biogenic reaction mediated by target microorganism.