Bioassay-guided isolation studies on the root extract of Polyalthia longifolia var. pendula possessing significant antibacterial activity led to the isolation of three new alkaloids pendulamine A (1), pendulamine B (2) and penduline (3) along with stigmasterol 3-O-beta-D-glucoside, allantoin, the known diterpenoid kolavenic acid, and the azafluorene alkaloid isoursuline. The structures of these compounds have been elucidated with the help of spectroscopic studies including 2D NMR experiments. Compounds 1 and 2, which are the only protoberberine alkaloids having a monosubstituted A ring with a hydroxy group at C-3, were found to be the active antibacterial principles of the roots. Their MIC values ranged between 0.02-20 microg against the tested bacteria.