Chagas disease is caused by the eukaryote parasite Trypanosoma cruzi. Current treatment exhibits limited efficacy and selenium-based compounds emerged as promising candidates for new therapies which is surpassing its bioisoster, sulfur. We designed new thiosemicarbazones, thiazoles, selenosemicarbazones and selenazoles, using isosteric substitution. We synthesized 57 new chalcogen compounds which were evaluated against T. cruzi, C2C12 cells and cruzain, the main target of this parasite. Additionally, human cathepsin L, was tested for selectivity. Three compounds were selected, based on their activity against the intracellular amastigotes (EC50 < 1 μM, SI > 10) and cruzain (IC50 < 100 nM, SI > 5.55) which compared favorably with the approved drug, Benznidazole, and the well-established cruzain inhibitor K777. Seleno-compounds demonstrated enhanced activity and selenazoles showed a decrease in selenium-associated toxicity. Compound 4-methyl-2-(2-(1-(3-nitrophenyl)ethylidene)hydrazineyl)-1,3-selenazole (Se2h) emerged as a promising candidate, and its binding to cruzain was investigated. Pharmacokinetic assessment was conducted, showing a favorable profile for subsequent in vivo assays.