Congenital infections by Trypanosoma cruzi and Toxoplasma gondii pose significant clinical challenges due to the lack of safe and effective treatments. This study evaluates eight novel 6-nitrocoumarin-3-thiosemicarbazone derivatives in an ex vivo human placenta model, assessing their antiparasitic activity and impact on tissue integrity. Two therapeutic approaches were tested: pre-infection (preventive) and post-infection (therapeutic). In vitro and ex vivo assays revealed strong activity trends. Compound 7 was the most effective against T. cruzi (IC50 = 22.4 ± 0.8 μM, logP = 2.49), while compound 1 exhibited the highest activity against T. gondii (IC50 = 17.3 ± 0.5 μM, logP = 1.44). Unlike current treatments, none of the compounds induced placental tissue damage, preserving trophoblast function. Structure-activity relationship (SAR) analysis identified an inverse correlation between lipophilicity and antiparasitic activity in T. gondii, where polar compounds were more effective. In T. cruzi, higher lipophilicity favored trypanocidal activity, suggesting differential cell permeability mechanisms. Mechanistic studies using electrochemistry and electron spin resonance (ESR) demonstrated that nitro group bioreduction promotes ROS generation, explaining activity against T. cruzi. By contrast, lower ROS levels in T. gondii suggest alternative mechanisms. This study validates the ex vivo human placenta model as a clinically relevant platform for antiparasitic drug screening. The findings highlight 6-nitrocoumarin-3-thiosemicarbazones as promising early-stage candidates that warrant further optimization to develop safer and more effective therapies for congenital infections.