The drugs used to treat cutaneous leishmaniasis (CL) cannot effectively penetrate lesions. Nanogold and nanosilver have been used for treating or enhancing drug delivery in CL. The present study used Commiphora molmol (myrrh) to synthesize silver nanoparticles (MSNPs). The MSNPs were characterized using transmission electron microscopy and energy-dispersive spectroscopy. In addition, antiparasitic effect of myrrh silver nanoparticles (MSNPs) was assessed on Leishmania major both in vitro and in vivo. Five concentrations of MSNPs (10, 50, 80, 100, and 150 μl/100 μL) were used to study their effect on L. major cultures in vitro, and MSNPs were also applied topically to subcutaneous lesions in mice in vivo. The results showed that the MSNPs were 49.09 nm in size. MSNPs, showed a marked and significant (p ≤ 0.05) growth inhibition of L. major promastigotes which was concentration dependent. Overall, the higher concentrations (100, 150 μl/100 μL had a significantly greater inhibitory effect for the MSNPs in comparison to the chemical nanoparticles (CNPs) and pentostam at the same concentrations. Lesions healed completely in 21 d after MSNP treatment in vivo, while pentostam, a commercial drug, and CNPs showed a moderate healing effect on the lesions. Thus, MSNPs were more effective than pentostam and CNPs both in the in vivo and in vitro studies. MSNPs can therefore be promising candidates for various nanomedicine applications.