Due to limited efficacy and considerable toxicity, the therapy for Chagas’ disease is far from being ideal, and thus new compounds are desirable. Diamidines and related compounds such as arylimidamides have promising trypanocidal activity against Trypanosoma cruzi. To better understand the mechanism of action of these heterocyclic cations, we investigated the kinetoplast DNA (kDNA) binding properties and trypanocidal efficacy against T. cruzi of 13 compounds. Four diamidines (DB75, DB569, DB1345, and DB829), eight arylimidamides (DB766, DB749, DB889, DB709, DB613, DB1831, DB1852, and DB2002), and one guanylhydrazone (DB1080) were assayed in thermal denaturation (Tm) and circular dichroism (CD) studies using whole purified T. cruzi kDNA and a conserved synthetic parasite sequence. The overall CD spectra using the whole kDNA were similar to those found for the conserved sequence and were indicative of minor groove binding. Our findings showed that some of the compounds that exhibited the highest trypanocidal activities (e.g., DB766) caused low or no change in the Tm measurements. However, while some active compounds, such as DB766, induced profound alterations of kDNA topology, others, like DB1831, although effective, did not result in altered Tm and CD measurements. Our data suggest that the strong affinity of amidines with kDNA per se is not sufficient to generate and trigger their trypanocidal activity. Cell uptake differences and possibly distinct cellular targets need to be considered in the final evaluation of the mechanisms of action of these compounds.