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Entamoeba histolytica is a cosmopolitan protozoan parasite that can produce infections in the intestine and some organs (liver, lungs, and brain), with worldwide prevalence. There are treatments against E. histolytica (antiparasitics), but as the drugs used in these treatments have presented some type of resistance and/or side effects, there are cases with complications of this disease. Therefore, it is necessary to develop new drugs aimed at a specific therapeutic target against this parasite. Here, we used the compound 5,5′‐[(4‐nitrophenyl)methylene]bis(6‐hydroxy‐2‐mercapto‐3‐methyl‐4(3H)‐pyrimidinone) in the patenting process (called D4). D4 has a reported specific use against a glycolytic enzyme, the triosephosphate isomerase of Trichomonas vaginalis (TvTIM). We determined that D4 has an amoebicidal effect in in vitro cultures, with an IC50 value of 18.5 µM, and we proposed a specific site of interaction (Lys77, His110, Gln115, and Glu118) in the triosephosphate isomerase of E. histolytica (EhTIM). Furthermore, compound D4 has favorable experimental and theoretical toxicity results. Therefore, D4 should be further investigated as a potential drug against E. histolytica.