Medicinal plants are widely used to treat disease in nonindustrialized societies for not being far cheaper than modern medicines. In this study, five types of medicinal plants viz., Glycyrrhiza glabra, Aegle marmelos, Centella asiatica, Cinnamomum verum, Cynodon dactylon were collected from Alwarkurichi, Tirunelveli District, Tamil Nadu, and extracts were prepared using various solvents such as methanol, ethanol, chloroform, diethyl ether, benzene, and water. These extracts were checked for antibacterial activity against Staphylococcus aureus isolated from a hospital environment. On the basis of antibacterial activity, three plants viz., Glycyrrhiza glabra, Aegle marmelos, and Centella asiatica were chosen for further research in which the ethanolic extract of Glycyrrhiza glabra rendered higher activity against Staphylococcus aureus. Thin Layer Chromatography of the plant extract of Glycyrrhiza glabra was carried out, which showed the development of four bands of which the first three bands rendered higher sensitivity against Staphylococcus aureus. The eluted bands from TLC were visualized under the UV-Spectrophotometer to monitor the absorbance of the eluted three bands in the wavelength spectrum ranging from 350-380 nm. Further confirmation of the separated compound was done by treating the band with Iodine and Ninhydrin spray for effective visualization of the isolated compound. The selected three bands from Glycyrrhiza glabra were taken for HPLC to quantify each component in the samples. Two to three clear peaks were obtained from the bands in HPLC indicates the presence of antibacterial activity against S. aureus.