Using a multimethod approach, including polarized light microscopy (PLM), scanning electron microscopy (SEM) with X-ray energy dispersive spectroscopy (EDS), SEM with mineral liberation analyzer (MLA), X-ray diffraction (XRD), and Raman spectroscopy, we examined sub-recent ferruginous crusts in the Xingu River in the Amazon Basin that have formed since the Early Pleistocene (<1.2 Ma), as indicated by (U-Th)/He dating of goethite. Although now preserved as goethite, the size and form of the smallest components of the ironstone (nanorods) and the nature of isomorphic substitution in the goethite point to very early transformation of the original precipitate, an unstable hydrous ferrous oxide (HFO) mineral, into goethite. The fine, multiply undulating laminae of the ironstone contain abundant filamentous microbial molds and casts that together support identification of the crusts as ferruginous microbialites and suggest a role of bioinduction/bioinfluence in ironstone precipitation, although inorganic precipitation is also evident. The Xingu Quaternary ironstones are the first evidence of ferruginous microbialite in a modern freshwater system in South America and may hold clues to the recent history of the Xingu River.