We have previously reported that epimastigote forms of Trypanosoma cruzi treated with the lysophospholipid analogues (LPAs) edelfosine (ET-18), ilmofosine, and miltefosine suffered alterations in plasma membrane, mitochondrion, and lipid synthesis. In this work, ET-18 induced membrane damage in trypomastigotes and amastigotes. Incubation of epimastigotes and trypomastigotes with the three LPAs led to membrane permeabilization, which was abolished by serum addition. Treatment for 24 h in culture medium interfered the with mitochondrial status of epimastigotes, with no effect in trypomastigotes, in agreement with ultrastructural data. LPAs induced alterations in the plasma membrane of the three forms of T. cruzi and in the mitochondria of epimastigotes, suggesting that these organelles are potential targets of these analogues.