Fast centrifugal partition chromatography (FCPC) was successfully applied in the separation of close Rf complex bioactive iridoid, loganetin directly from the ethyl acetate extract of Alstonia scholaris. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)/ACN/Water (3:1.5:3v/v/v) where the lower phase of the biphasic system, the aqueous layer containing 8mM HCl, was the stationary phase, while the upper organic layer supplemented with 15mM triethylamine TEA was designated as the mobile phase. From 1.5g of EtOAc extract, 48mg of loganetin (1) was obtained in 94.4% purity as determined by HPLC. The isolated compound (1) was characterized on the basis of its (1) H, (13) C-NMR, and ESI-MS spectroscopic data. Although loganetin does not possess antibacterial activity of its own, but in combination, it appreciably reduces the minimum inhibitory concentration (MIC) of nalidixic acid (NA) against nalidixic acid resistant (NAREC) and nalidixic acid sensitive (NASEC) strains of Escherichia coli. Loganetin, a very common, inexpensive, and non-toxic natural product may finds its application in the antibacterial drug development for treating multidrug-resistant Gram negative infections.