Current treatment for leishmaniasis relies on highly toxic drugs that cause undesirable side effects. Therefore, the search for new drugs with high leishmanicidal activity and reduced toxicity is crucial. This study assesses the in vitro anti-leishmanial activity of Diethyldithiocarbamate (DETC), a copper chelator, and D81, an inorganic compound with an amine group. The combination of these drugs targets Leishmania (L.) braziliensis, L. amazonensis, and L. infantum by inhibiting SOD1 and increasing superoxide and nitrite peroxide production. Toxicity tests were conducted on the promastigotes and amastigotes of the mentioned Leishmania species, and murine macrophages derived from BALB/C mice. The 50% inhibition index was 33.03μM for D81, 0.247μM for DETC, and 1.676μM for the combination (1:2.5), with similar results for all three species. The inhibition index for murine macrophages was 237μM for D81, 11.47μM for DETC, and 67.16μM for the combination (1:2.5). The combined use of 20μM DETC and 500μM D81 demonstrated an antagonistic action, resulting in no inhibition of cell viability in murine macrophages, indicating less toxicity than the individual drugs. Conversely, the combination resulted in a synergistic effect and complete inhibition of cell viability in Leishmania species. The combination of DETC and D81 reduced the infection rate by over 44% and the number of amastigotes per 100 cells by more than 77% in murine macrophages infected by L. braziliensis, L. amazonensis, and L. infantum, compared to the control group. Preliminary results also showed a similar reduction in human macrophages infected with L. infantum. In conclusion, the association of D81 with DETC demonstrated potent antiproliferative effects on promastigotes and amastigotes of L. braziliensis, L. amazonensis, and L. infantum, with reduced toxicity in murine macrophages. Further studies are needed to investigate its effect on human macrophages infected by L. braziliensis and L. amazonensis.