Iodinated X-ray contrast media (ICM) are commonly detected in the aquatic environment at concentrations up to the low microgram per liter range. In this study, the biotransformation of selected ICM (diatrizoate, iohexol, iomeprol, and iopamidol) in aerobic soil-water and river sediment-water batch systems was investigated. In addition, microbial transformation pathways were proposed. Diatrizoate, an ionic ICM, was not biotransformed, while three nonionic ICM were transformed into several biotransformation products (TPs) at neutral pH. Iohexol and iomeprol were biotransformed to eleven TPs and fifteen TPs, respectively, while eight TPs were detected for iopamidol. Since seven of the TPs detected during biotransformation had not been previously identified, mass fragmentation experiments were completed to elucidate the chemical structures. Oxidation of primary alcoholic moieties, cleavage of the N-C bonds (i.e., deacetylation and removal of hydroxylated propanoic acids), and decarboxylation are potential reactions that can explain the formation of the identified TPs. Iohexol and iomeprol had similar biotransformation rates, while iopamidol was biotransformed slower and to a lesser extent. A LC tandem MS method confirmed the presence of ICM TPs in aqueous environmental samples. Fifteen of the ICM TPs were even detected in drinking water with concentrations up to 120 ng/L.