The present work assesses the effect in vitro of combining the antiretroviral drug nelfinavir (NFV), a drug used against HIV but also a strong in vitro inhibitor of the growth of Leishmania promastigotes and amastigotes, with amphotericin B or miltefosine on different strains of Leishmania infantum. The isobolograms revealed a synergistic effect for both combinations, with a fractional inhibitory concentration index (∑FIC) <0.5. The ∑FIC values obtained with reference strain MCAN/ES/98/LLM-724 were 0.25 ± 0.1 (95% CI: 0.178-0.322) for the combination NFV+AMB and 0.48 ± 0.2 (95% CI: 0.377-0.573) for the combination NFV+MTF. The effect of NFV on visceral leishmaniasis induced by L. infantum in BALB/c mice was also examined, and the results confirmed a leishmanicidal effect when administered alone, with approximately 60% (P<0.05) reductions in the liver and spleen parasite burdens. This is the first time this has been reported in an in vivo model. A significant reduction in the liver (77%; P<0.01) and spleen (76%; P<0.01) parasite burdens was also observed for NFV+MTF compared with those obtained when these drugs were used alone. This indicates that such combinations may be useful treatment options in patients with visceral leishmaniasis who are also infected with HIV.