The toxicity and antileishmanial activity of 20 betulin derivatives were studied. The toxicity of betulin and synthesized compounds was determined using a bacterial test (Microtox) and two mammalian cell lines (CHO-K1 and J774). The antileishmanial activity of compounds (50 μM) was examined in both the promastigote and intracellular amastigote stages of Leishmania infantum and L. donovani. No correlation was found among the toxicity tests. All the compounds showed significant antipromastigote activity. The antiproliferative capacity of derivatives was dependent on the parasite stage studied, and no substantial differences were found between Leishmania species. Betulin, 3,28-di-O-acetylbetulin and L-aspartyl amide of betulonic acid showed moderate activity against amastigotes. The highest inhibition of intracellular amastigote multiplication was achieved with a low micromolar concentration (IC50 ca 9 μM) of heterocyclic betulin derivative 3,28-di-O-acetyllup-13(18)-ene with N-ethyltriazolo moiety 16, without significant toxicity for mammalian cells. These results point to the interest of this lead compound for further in vitro and in vivo tests.