Therapeutic potential of Gymnema sylvestre on diverse cell types is predominantly due to a variety of terpenoids and their derivatives. However, their bioavailability becomes limited due to poor solubility and lower lipophilic properties, provoking the search for novel membranotropic terpenoids and their mechanism of action. A terpenoid fraction purified from Gymnema sylvestre exhibited broad spectrum antimicrobial activity against both Gram positive and Gram negative bacteria with IC50<0.1 mg/ml. Evaluation of its membranotropic effect in vitro on reconstituted model membrane revealed that the fraction induced flip-flop of fluorescent phospholipid analogs across the lipid bilayer. The terpenoid-induced lipid flipping was biphasic with a fast linear phase (rate constant (k(1)) = 3 to 5 S-1) and a second slow exponential phase (rate constant (k(2)) = (4 to 9) x 10(-3) S-1). The lipid-flippase activity of the terpenoid fraction showed concentration and incubation-dependent cooperativity, indicating their lipophilic nature and membrane-destabilizing activity that facilitated lipid translocation. For the first time, our study reveals the flippase activity of a terpenoid fraction of Gymnema sylvestre that could be further explored for their membrane-mediated pharmacological properties.