Entamoeba histolyticais a disease-causing parasitic amoeba which affects an estimated 50 million people worldwide, particularly in socioeconomically vulnerable populations experiencing water sanitation issues. Infection withE.histolyticais referred to as amoebiasis, and can cause symptoms such as colitis, dysentery, and even death in extreme cases. Drugs exist that are capable of killing this parasite, but they are hampered by downsides such as significant adverse effects at therapeutic concentrations, issues with patient compliance, the need for additional drugs to kill the transmissible cyst stage, and potential development of resistance. Past screens of small and medium sized chemical libraries have yielded anti-amoebic candidates, thus rendering high-throughput screening a promising direction for new drug discovery in this area. In this study, we screened a curated 81,664 compound library from Janssen pharmaceuticals againstE.histolyticatrophozoitesin vitro, and from it identified a highly potent new inhibitor compound. The best compound in this series, JNJ001, showed excellent inhibition activity againstE.histolyticatrophozoites with EC50values at 0.29 μM, which is better than the current approved treatment, metronidazole. Further experimentation confirmed the activity of this compound, as well as that of several structurally related compounds, originating from both the Janssen Jump-stARter library, and from chemical vendors, thus highlighting a new structure-activity relationship (SAR). In addition, we confirmed that the compound inhibitedE.histolyticasurvival as rapidly as the current standard of care and inhibited transmissible cysts of the related model organismEntamoeba invadens. Together these results constitute the discovery of a novel class of chemicals with favorablein vitropharmacological properties. The discovery may lead to an improved therapy against this parasite and in all of its life stages.