Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in the Trypanosoma cruzi glycolytic pathway, parasite that causes Chagas' disease. There are only few drugs available to treat this disease, most of which present strong side effects. Natural products constitute a prime source for the discovery of new scaffolds potentially useful for the treatment of several diseases, including Chagas' disease. Bioactivity-guided fractionation of Spiranthera odoratissima extract using T. cruzi GAPDH (TcGAPDH) as a target led to the isolation of the flavonoid tiliroside (kaempferol-3-O-β-D-(6''-trans-p-coumaroyl)-glucopyranoside), identified as an excelent inhibitor of this enzyme and for the first time reported for this plant species. Mechanistic studies of tiliroside showed that it is a reversible non-competitive inhibitor of TcGAPDH. In additon, molecular modeling analysis indicated the binding mode of tiliroside to TcGAPDH. Therefore, the identification of tiliroside as TcGPADH inhibitor in a complex matrix such as the plant crude extract and the discovery of a new binding site may contribute to the opening of new paths in the search for natural product inhibitors of this enzyme.