Although aerobic degradation of ethylene glycol is well documented, anaerobic biodegradation only via methanogenesis or fermentation has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of shallow-rooted plants such as alfalfa and grasses where conditions can be aerobic, but it has not been demonstrated in the deeper rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol degradation under nitrate-, and sulfate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees planted in the path of a groundwater plume containing up to 1.9 mol/L (120 g/L) ethylene glycol and the effect of fertilizer addition when nitrate or sulfate was provided as a terminal electron acceptor (TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulfate as TEAs at close to the theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulfate was used primarily as TEA, TEAs naturally present in the soil or carbon dioxide produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster, close to 100% disappearance eventually was achieved anaerobically. Degradation rates under nitrate-reducing conditions were enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene glycol degradation/L/day in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep-rooted trees like willow and poplar can degrade ethylene glycol anaerobically. If anaerobic biodegradation contributes significantly to the phytoremediation of ethylene glycol in the deeper subsurface, the need for pump and treat or aerobic treatment might be eliminated, thereby reducing the cost of treatment.