Tinospora cordifolia is an herbaceous vine of the family Menispermaceae. This plant is indigenous to the tropical areas and distributed throughout India, Myanmar and Sri Lanka. Phytochemicals investigation revealed crude and treated extracts of the T. cordifolia plants stem were contain more or less same type of chemical constituents using extract methanol, chloroform and petroleum ether. This plant stem extracted from various organic solvent were tested against some Gram-positive, Gram-negative bacteria and fungi. Selected human pathogens Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli and Fungi .The total eight samples of the five bacteria were study for antibiotic susceptibility against standard antibiotics like Gentamicin. Antibacterial activity of crude extract was carried out using disc diffusion method and MIC of isolated compound was tested by broth microdilution method. Thus the present study suggested crude petroleum ether extract showed good antibacterial activity against tested microbes.