Neglected parasitic diseases as Leishmaniasis and Chagas disease affect 12 and 6 million people in tropical and subtropical countries, respectively. Co-infections with HIV and other parasitic diseases represent a major threat to Public Health. With a limited number of drugs in clinical use, the treatment is still deficient, including severe adverse effects [1]. Guanidine compounds has been widely found in natural products and especially in marine invertebrates. Marine sponges, in particular, are by far the most abundant source of all natural guanidine derivatives; the structural complexity of these compounds and their potent biological activities make these compounds a target of choice for studies in organic synthesis [2]. In this work, we studied the in vitro antileishmanial activity of a synthetic batzelladine alkaloid derivative and a ptilomycalin A derivative and their capability to alter the permeability of the plasma membrane of Leishmania. The in vitro assays demonstrated that Leishmania (L.) infantum was susceptible to the guanidine derivatives, EB-3 and GB-118, with IC50 values of 57 µg/mL and 33 µg/mL, respectively. The compounds also eliminated the intracellular forms of Leishmania with an IC50 value of 5 µg/mL and 2 µg/mL, respectively. The cytotoxicity of both compounds against NCTC cells resulted in CC50 values of 137 µg/mL and 45 µg/mL, demonstrating selectivity indexes of 27 and 22, respectively. Using the fluorescent probe SYTOX Green, it was possible to demonstrate that both compounds altered the plasma membrane permeability of Leishmania at the early time of incubation (<60 min), but EB-3 affected the membrane to a higher extent, when compared to the positive control with Triton-X 100. These results indicate the potential of synthetic guanidine derivatives as novel drug prototypes for Leishmaniasis.