Despite the continuous research effort that has been made in recent years to find ways to treat the potentially life threatening Chagas disease (CD), this remains the third most important infectious disease in Latin America. CD is an important public health problem affecting 6-7 million people. Since the need to search for new drugs for the treatment of DC persists, in this article we present a panel of new polyamines based on the tripodal structure of tris(2-aminomethyl)amine (tren) that can be prepared at low cost with high yields. Moreover, these polyamines present the characteristic of being water-soluble and resistant to the acidic pH values of stomach, which would allow their potential oral administration. In vitro and in vivo assays permitted to identify the compound with the tren moiety functionalized with one fluorene unit (7) as a potential antichagas agent. Compound 7 has broader spectrum of action, improved efficacy in acute and chronic phases of the disease and lower toxicity than the reference drug benznidazole. Finally, the action mechanisms studied at metabolic and mitochondrial levels shows that the trypanocidal activity of compound 7 could be related to its effect at the glycosomal level. Therefore, this work allowed us to select compound 7 as a promising candidate to perform preclinical evaluation studies.