Herbal medicines have gained global importance, both medically as well as economically. Bacopa monnieri (L.) is commonly and widely called as brahmi, belongs to the family Scrophulariaceae. It is a traditional plant in India, which has been used for centuries to increase mental capacity, improve mental and brain functions. The plant was reported to have anti-inflammatory, analgesic, antipyretic, antioxidant and anticancer activities. In the present study, a comparative analysis of antioxidant and phytochemical content difference is done among the in vitro and in vivo cultivated plants of three different cultivars of Bacopa monnieri (L.). In DPPH free radical assay, cultivar procured from Chuharpur showed 95.47% of inhibition at 70µg/ml concentration whereas 95.02% of inhibition of nitric oxide (at conc.140µg/ml) in nitric oxide free radical assay. Callus and in vitro grown plants of all the three cultivars of Bacopa monnieri showed positive test for amino acids, carbohydrates, alkaloids, glycosides, flavonoids, phenolic compounds, carotenoids and terpenoids.