The parts of the Nicotiana tabacum plant have been known to possess a wide range of biological activities. The purpose of the present study was to investigate the antibacterial activity, phytochemical screening and the antioxidant activity of aqueous and alcoholic extracts of the stem of Nicotiana tabacum. The antibacterial activity was observed against two gram positive bacteria (Bacillus amyloiquefaciens, Staphylococcus aureus) & two gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa) in its aqueous, ethanolic, acetone and methanolic extracts by agar well diffusion method where maximum antibacterial activity found to be present in the methanolic and ethanolic extract against Staphylococcus aureus with an inhibition length of 10.667 +/- 1.527 mm and 8 +/- 1.00 mm, respectively. Phytochemical screening of aqueous, ethanolic & methanolic extract revealed the presence of saponin, flavonoids and alkaloids. Flavonoid content in stem was found to be 838 mg QE/g of extract in the 80% of ethanol extract by aluminium chloride colometric method. Antioxidant activity was observed in order to estimate the superoxide dismutase, catalase, glutathione content, glutathione s transferase & lipid peroxidation i.e. Malondialdehyde (MDA) content in the aqueous and methanolic extract where methanolic extract has shown a high level of antioxidant activity. The present study suggests that the stems of Nicotiana tabacum can be as a good antimicrobial and antioxidant agent.