Infectious diseases caused by microorganisms are one of the major causes of morbidity and mortality around the world. Emergence of new infectious strains and evolution of antibiotic resistance among them are the major threats the man kind is facing. This expands the need of discovery of newer and stronger antimicrobial compounds from the safer sources such as plants. Glinus oppositifolius (L.) Aug. DC. is a perennial herb which is predominantly being used in traditional medicine across different ethnicity. The plant extract has been reported to contain several medicinally important secondary metabolites; however, research on purified compounds from the plant is highly limited. This present study is focused to extract and partially purify flavonoid glycosides from G. oppositifolius and to evaluate its antibacterial efficiency. The aerial parts of G. oppositifolius were macerated and extracted and tested for presence of flavonoid glycosides which are potent anti-bacterial compounds. The quantitative estimation has shown 210.83 ± 4.63 mg of flavonoids and 167.77 ± 1.73 mg of carbohydrates per gram of dry extract. Further, partially purified flavonoid glycosides were isolated from the crude extract using silica gel column chromatography and quantitatively tested to contain 67.92 ± 0.58 mg of flavonoids and 38.54 ± 1.75 mg of carbohydrates per gram of dry fraction. The partially purified fractions were subjected to high-performance liquid chromatographic separation for further purification. In vitro antibacterial activity and minimum inhibitory concentration of the flavonoid glycosides were examined against six pathogenic bacteria through well diffusion and micro dilution methodologies. The flavonoid glycosides showed strong antibacterial activity against all bacteria tested. The concentration of 2800 μg/ml has registered 100% of inhibition against all bacteria tested. Conclusively, the results from the present study indicated that the methanolic extract of G. oppositifolius can yield good amount of flavonoid glycoside which shows strong and significant antibacterial activity and can be further explored for different medical applications.