The plants of genus Ammannia are widely used in traditional Chinese medicinal system but no detailed phytochemical investigations on A. multiflora have been carried so far. The aim of present study was to carry out chemical investigation of A. multiflora. In a preliminary screening, glycosides, flavonoids, carbohydrates, steroids, phenols and sterols were detected but alkaloids were absent. Further, plant material was successively extracted with hexane, chloroform and methanol and the chloroform extract was subjected for chromatographic separation over Vacuum Liquid Chromatography (VLC). The non-polar VLC fractions of chloroform extract along with hexane extract were later analyzed with GC-MS which resulted in the characterization of fourteen constituents being reported for the first time from A. multiflora. Some of the constituents were potentially active and could be used in future development of anti-microbial agents.