The interaction of miltefosine with amphotericin B, sodium stibogluconate, paromomycin, and sitamaquine was assessed in vitro and additionally for the first three combinations in vivo. In vitro interactions were indifferent for miltefosine combined with amphotericin B (mean sums of fractional inhibitory concentrations [mean summation operatorFICs] ranging from 1.22 to 1.51 at the 50% effective concentration [EC50] level and 1.08 to 1.38 at the EC90 level), sitamaquine (mean summation operatorFICs from 1.33 to 1.38 and 1.0 to 1.02, respectively), and paromomycin (mean summation operatorFICs from 0.79 to 0.93 at the EC50 and 0.77 to 1.35 at the EC90 level). Some synergy was observed for miltefosine combined with sodium stibogluconate (mean summation operatorFICs from 0.61 to 0.75 at EC50 and 0.49 to 0.97 at EC90). Different interactions were found in vivo, where the highest potentiation of miltefosine activity was achieved with amphotericin B (activity enhancement index [AEI] of up to 11.3). No significant interaction was observed when miltefosine was combined with sodium stibogluconate (AEI of up to 2.38). The potentiation of miltefosine in vivo was also achieved with the combination of miltefosine and paromomycin (AEI of up to 7.22).