The present study was aimed to investigate the antimicrobial activity and characterization of bioactive constituents from flower extract of Cassia javanica Linn. Antimicrobial activity was tested against human pathogens in six different solvents using the well diffusion method. These, ethyl acetate and ethanolic crude extracts showed effective antimicrobial activity against all the tested human pathogens. The bioactive constituents of the flower extract revealed the presence of some biologically active substance alkaloids, cardiac glycosides, flavonoids, glycosides, phenolic compounds, phlobatannins, triterpenoides, saponins, volatile oils and tannins. In the GC-MS analysis, 14 bioactive compounds were identified. Present study concludes that the different extracts of Cassia. javanica Linn. Flower contain a broad spectrum of bioactive compounds and also exhibit antimicrobial activity against all the tested human pathogens, Therefore, Cassia. javanica Linn. Flowers can be tapped source to discover the natural bioactive products that may serve as leads in the developments of novel therapeutic drugs.