The antitrypanosomal activity of 10 already synthesized compounds was in silico predicted as well as in vitro and in vivo explored against Trypanosoma cruzi. For the computational study, an approach based on non-stochastic linear fingerprints to the identification of potential antichagasic compounds is introduced. Molecular structures of 66 organic compounds, 28 with antitrypanosomal activity and 38 having other clinical uses, were parameterized by means of the TOMOCOMD-CARDD software. A linear classification function was derived allowing the discrimination between active and inactive compounds with a confidence of 95%. As predicted, seven compounds showed antitrypanosomal activity (%AE > 70) against epimastigotic forms of T. cruzi at a concentration of 100 μg/mL. After an unspecific cytotoxic assay, three compounds were evaluated against amastigote forms of the parasite. An in vivo test was carried out for one of the studied compounds.