Background: Chagas disease is a major cause of morbidity and death for millions of people in Latin America. The drugs currently available exhibit poor efficacy and severe side effects. Therefore, there is an urgent need for new, safe and effective drugs against Chagas disease. The vital dependence on glycolysis as energy source makes the glycolytic enzymes of Trypanosoma cruzi, the causative agent of Chagas disease, attractive targets for drug design. In this work, glyceraldehyde-3-phosphate dehydrogenase from T. cruzi (TcGAPDH) was employed as molecular target for the discovery of new inhibitors as hits. Results: Integrated protein-based pharmacophore and structure-based virtual screening approaches resulted in the identification of three hits from three chemical classes with moderate inhibitory activity against TcGAPDH. The inhibitors showed IC₅₀ values in the high micromolar range. Conclusion: The new chemotypes are attractive molecules for future medicinal chemistry efforts aimed at developing new lead compounds for Chagas disease.