Several β-carboline compounds were evaluated for in vitro trypanocidal activity against Trypanosoma cruzi and their potential toxic effects was also assessed. β-Carboline derivative 4 showed good activity against epimastigote, trypomastigote, and amastigote forms of T. cruzi, with a dose-dependent inhibitory effect. It showed an IC 50 of 14.9 μM against the epimastigote form and an EC 50 of 45 μM and 33 μM against trypomastigote and amastigote forms, respectively. Additionally, 4 was able to be active on mammalian cell–protozoan interaction, reducing the number of infected cells and the number of internalized parasites. The compound showed low cytotoxicity, with a selective index 31 times higher to the parasite than for mammalian cells. In human red-blood cells β-Carboline 4 at 14.9 μM not caused haemolysis. Observed at electron microscopy 4-treated epimastigotes showed abnormal swelling of the mitochondrion, a diffuse kinetoplast, and distortions of the parasite cell body. The present data support the potential effect of this class of compounds against T. cruzi and encourage further experiments in vitro to evaluate the action mechanism of this drug and also with in vivo models.