A series of bis-(arylmethylidene)-cycloalkanones was synthesized by cross-aldol condensation. The activity of the compounds was evaluated against amastigotes forms of Trypanosoma cruzi and promastigotes forms of Leishmania amazonensis. The cytotoxicity of the active compounds on uninfected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their antiparasitic effects. Six compounds displayed trypanocidal activity against amastigotes intracellular forms of T. cruzi with IC50 values ranging from 7.0 to 249 μM. Besides these six compounds, eight other molecules exhibited significant leishmanicidal activity (IC50 values ranging from 0.6 to 110.4 μM). Two compounds can be considered as promising antiparasitic lead molecules because they showed IC50 values in the low-micromolar range (≤1.2 μM) with an adequate SI (≥19.9). To understand the mechanism of action of these compounds, two possible molecular targets were investigated: trypanothione reductase (TR) and cruzain.