The formation of water-stable aggregates in finely textured and polymineral magnetite Fe ore tailings is one of the critical processes in eco-engineering tailings into soil-like substrates as a new way to rehabilitate the tailings. Organic matter (OM) amendment and plant colonization are considered to be effective in enhancing water-stable aggregation, but the underlying mechanisms have not yet been elucidated. The present study aimed to characterize detailed changes in physicochemistry, Fe-bearing mineralogy, and organo-mineral interactions in magnetite Fe ore tailings subject to the combined treatments of OM amendment and plant colonization, by employing various microspectroscopic methods, including synchrotron-based X-ray absorption fine structure spectroscopy and nanoscale secondary ion mass spectroscopy. The results indicated that OM amendment and plant colonization neutralized the tailings' alkaline pH and facilitated water-stable aggregate formation. The resultant aggregates were consequences of ligand-promoted bioweathering of primary Fe-bearing minerals (mainly biotite-like minerals) and the formation of secondary Fe-rich mineral gels. Especially, the sequestration of OM (rich in carboxyl, aromatic, and/or carbonyl C) by Fe-rich minerals via ligand-exchange and/or hydrophobic interactions contributed to the aggregation. These findings have uncovered the processes and mechanisms of water-stable aggregate formation driven by OM amendment and plant colonization in alkaline Fe ore tailings, thus providing important basis for eco-engineered pedogenesis in the tailings.