The severe toxicity, exorbitant cost and the emerging resistance of Leishmania spp. against most of the currently used drugs led to the urgent need for exploiting our traditional Ayurvedic knowledge to treat visceral leishmaniasis. The aim of this study was to evaluate the in vitro anti-leishmanial activity of various extracts from ten traditionally used Indian medicinal plants. The methanolic extract from only two plants, Withania somnifera Dunal (ashwagandha) and Allium sativum Linn. (garlic), showed appreciable activity against Leishmania donovani. Further active compounds from these two plants were isolated and purified based on bioactivity-guided fractionation. HPLC-purified fraction A6 of ashwagandha and G3 of garlic showed consistently high activity with 50% inhibitory concentration (IC(50)) of 12.5 +/- 4 and 18.6 +/- 3 microg/ml against promastigotes whereas IC(50) of 9.5 +/- 3 and 13.5 +/- 2 microg/ml against amastigote form, respectively. The fraction A6 of ashwagandha was identified as withaferin A while fraction G3 of garlic is yet to be identified, and the work is in progress. Cytotoxic effects of the promising fractions and compounds were further evaluated in the murine macrophage (J774G8) model and were found to be safe. These compounds showed negligible cytotoxicity against J774G8 macrophages. The results indicate that fraction A6 of ashwagandha and fraction G3 of garlic might be potential sources of new anti-leishmanial compounds. The in vivo efficacy study and further optimization of these active compounds are in progress.