Deep subsurface oxic/anoxic interfaces within Henderson Mine, CO were investigated for the potential to support novel metal-oxidizing microorganisms. Ralstonia sp. were isolated from Fe-oxidizing enrichments inoculated with fracture fluids released through boreholes as well as Fe-oxides mineralizing around the mouths of the boreholes. 16S rRNA clone libraries of environmental DNA revealed that closely related Ralstonia sp. were numerically-dominant in metal-rich subsurface fluids. FeCO3 gradient tubes were then utilized to demonstrate that isolate Ralstonia HM08-01 grows by oxidizing Fe(II) with O2 at circumneutral pH. These results and the geochemical data from the borehole fluids implicate Fe-oxidation as a viable subsurface lifestyle. The differential development of Fe-oxide bands in biotic vs. abiotic gradient tubes suggests that Ralstonia HM08-01 exerts spatial control over Fe oxidation and precipitation. Geochemical profiles of Fe(II), Fe(III) and O2 taken through the gradient tubes with voltammetric microelectrodes reveal that despite visual differences, similar total concentrations and distributions of aqueous Fe species were present in both systems. Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used to characterize the mineralogy of the Fe-oxides produced in biotic vs. abiotic experiments. 2L-ferrihydrite dominated the mineral fits in both systems, and SEM revealed the ferrihydrite particles to be 50–100 nm in diameter. This mineralogical identification combined with the detection of an abundant electroactive Fe(III) species are used to infer that 2L-ferrihydrite is a long-term stabilized colloidal species. The mechanism for stabilization of this phase is the presence of PO42− and Si in growth experiments. In the Henderson fluids, PO42− is below detection, but Si is at micromolar concentrations and likely influences the formation of potentially colloidal Fe-oxides in the environment.