Rio Grande Rise (RGR) is an intraplate oceanic elevation in the South Atlantic Ocean that formed at a hotspot on the Mid-Atlantic Ridge during the Cretaceous. In spreading center and hotspot environments, ironstones form mainly by biomineralization of reduced Fe from hydrothermal fluids or oxidation of sulfide deposits. However, RGR has been considered aseismic and volcanically inactive for the past 46 Ma. Here, we investigate the origin of ironstones collected from the summit of RGR using multiple techniques: petrographic observations, X-ray diffraction, U-Th/He geochronology, and chemical composition. The ironstones from RGR consist of finely laminated goethite containing igneous rock fragments, carbonate fluorapatite, and calcite. Our results suggest that Fe oxyhydroxides were precipitated by Fe-oxidizing bacteria forming bacterial mats. The bacterial Fe mats underwent compaction, dewatering, goethite crystallization, and cementation that created the ironstone deposits. U-Th/He geochronology reveals protracted goethite minimum ages extending from the late Miocene to the Quaternary, probably due to multiple generations of mats, slow mineralization rates, and Fe-oxide dissolution-reprecipitation cycles. Flame-like goethite structures underneath FeMn crusts and a chimney-shaped goethite sample with a central channel indicate that the dewatering fluid flowed upward through the deposits, or a thermal fluid source may have been introduced from below the ironstone deposits. High Fe/Mn ratios, low trace metals contents (Ni + Co + Cu), and very low Fe/REY ratios suggest ironstone precipitation from a hydrothermal fluid; however, REY_SN plots and bivariate Ce_SN /Ce_SN^⁎ versus Y_SN/Ho_SN and Ce_SN /Ce_SN^⁎ versus Nd plots are inconclusive, and a proximal source of magma was unlikely during the period of mat formation. Given this evidence, we hypothesize that a geothermal circulation system may have facilitated ironstone mineralization at RGR.