Growth inhibitory activity in vitro of sixteen new 5-nitrofuryl derivatives against the protozoan parasite Trypanosoma cruzi, the causative agent of American trypanosomiasis, was studied. The designed compounds combine in the same molecule the recognized 5-nitrofuryl group, an oxidative stress promoter, and lateral chains that could interact with biomolecules such as trypanothione reductase. Some of the derivatives were found to be very active against the epimastigote form of the parasite, being near to 3.0-fold more active than the reference compound, nifurtimox. Moreover, three-dimensional requirements for activity were clearly observed using a 3D-QSAR study based on a comparative molecular field analysis (CoMFA). The best CoMFA model, r(2) = 0.970 and q(2) = 0.725, points to the importance of a specific hydrogen-bonding pattern around the carbonyl or thiocarbonyl moieties, as well as the requirement for hydrophobic lateral chains. Theoretical pharmacokinetics (Lipinski's rule, PSA) supports further in vivo studies.