BACKGROUND & OBJECTIVES The severe toxicity, exorbitant cost and emerging resistance of Leishmania species against most of the currently used drugs underscores the urgent need for the alternative drugs. The present study evaluates in vitro anti-leishmanial activity of Plumeria bicolor and its isolated compounds. METHODS The in vitro anti-parasitic activity of chloroform extract of Plumeria bicolor, plumericin and isoplumericin were tested alongwith appropriate controls against promastigote and amastigote forms of Leishmania donovani using 96 well microtiter plate. The concentration used for assessing the anti-leishmanial activity of extract of Plumeria bicolor and both isolated compounds were 100 μg/ml and 15 μM, respectively. The viability of the cells was assessed by MTT assay. The cytotoxicity of these compounds was performed against J774G8 murine macrophage cells lines at the concentration of 30 μM. RESULTS The Plumeria bicolor extract showed activity with the IC 50 of 21±2.2 and 14±1.6 μg/ml against promastigote and amastigote forms of L. donovani, respectively. Plumericin consistently showed high activity with the IC 50 of 3.17±0.12 and 1.41±0.03 μM whereas isoplumericin showed the IC50 of 7.2±0.08 μM and 4.1±0.02 μM against promastigote and amastigote forms, respectively. Cytotoxic effect of the chloroform extract of P. bicolor, plumericin and isoplumericin was evaluated in murine macrophage (J774G8) model with CC50 value of 75±5.3 μg/ml, 20.6±0.5 and 24±0.7 μM, respectively. INTERPRETATION & CONCLUSIONS Our results indicated that plumericin showed more potent activity than isoplumericin and might be a promising anti-leishmanial agent against L. donovani.