The oxygenation of Earth’s atmosphere allowed for the diversification of metabolisms to include those that rely on oxygen and its derivatives. For example, chemolithotrophic oxidation of sulfide and iron both require oxygen or nitrate as terminal electron acceptors. A growing number of oxygen-utilizing chemolithotrophs are known to accumulate intracellular polyphosphate as an energy reserve that allows them to adapt to the fluctuating redox conditions in their distinctive-gradient habitats. Polyphosphate is also thought to play an important role in the formation of phosphatic mineral deposits. Here we present fossil evidence of iron-oxidizing bacteria preserved as filamentous iron oxides within phosphatic Paleoproterozoic stromatolites. The filaments include twisted stalks similar to those produced by modern iron-oxidizing bacteria that are known to metabolize polyphosphate and inhabit steep redox gradients. Fossil iron-oxidizing bacteria preserved within some of the oldest known phosphorites serve as indicators of O2-Fe(II) gradients that may have supported microbially mediated phosphogenesis via polyphosphate metabolism and/or an active iron redox pump.