ABSTRACT Current monotherapy against visceral leishmaniasis has serious side effects, and resistant Leishmania strains have been identified. Amphotericin B (AmB) has shown an extraordinary antileishmanial efficacy without emergence of resistance; however, toxicity has limited its general use. Results obtained showed, using a fixed-ratio analysis, that the combination of diallyl thiosulfinate (allicin) and AmB ranged from moderately synergic to synergic at low concentrations (0.07 μM AmB plus 35.45 μM allicin induced 95% growth inhibition). None of the treatments, alone or in combination, had noticeable adverse effects on macrophages (Mϕ) in the concentration range examined (allicin, 0.5, 1, 5 and 10 μM; AmB, 0.05, 0.075, and 0.1 μM). Allicin, AmB, or the combination did not affect the infection rate (percentage of infected Mϕ) of Leishmania . Allicin enhanced the activity of AmB on intracellular amastigotes of Leishmania donovani and L. infantum (ca. 45% reduction of amastigote burden with 0.05 μM AmB plus 10 μM allicin); this represented nearly a 2-fold reduction in the 50% inhibitory concentration (IC 50 ) of the antibiotic added alone. Results point toward the possible utility of testing this combination in vivo to reduce the toxicity associated with monotherapy with AmB.