Glycerol trinitrate (GTN) is a contaminant commonly found in the waste streams of munitions manufacturing and pharmaceutical plants. It is a highly toxic substance harmful to humans and other living organisms. In this study, the bioconversion of GTN was examined under aerobic conditions using mixed bacterial cultures and Phanerochaete chrysosporium, and under strict anaerobiosis using anaerobic digester sludge. Batch reactor experiments indicated that activated sludge, P. chrysosporium, and anaerobic digester sludge can completely denitrate GTN via a mechanism which forms isomeric glycerol dinitrate (GDN), glycerol mononitrate (GMN) and a utilizable carbon source, most likely glycerol. The rate of bioconversion in the aerobic and the anaerobic systems was dependent upon the concentration of cosubstrate. Continuous flow experiments in immobilized bed reactors indicated that anaerobic degradation of GTN can achieve high destruction efficiencies of the parent compound (99.9%) and the intermediate metabolic nitrate ester compounds (GDN, GMN) at relatively low cosubstrate requirements compared to the aerobic reactors.