The total phenolic and flavonoid content and percentage of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of callus and in vivo plant parts of Heliotropium indicum Linn. were estimated. Murashige and Skoog (MS) basal medium supplemented with alpha-naphthaleneacetic acid (NAA) 2.0 mg/l with benzyladenine (BA) 0.5 mg/l showed the highest amount of callus biomass (1.87 g/tube). The morphology of callus was significantly different according to the plant growth regulators and their concentrations used in the medium. The highest amount of total phenolic (21.70 mg gallic acid equivalent per gram (GAE/g)) and flavonoid (4.90 mg quercetin equivalent per gram (QE/g)) content and the maximum percentage (77.78 %) of radical scavenging activity were estimated in the extract of inflorescence. The synergistic effect of NAA (2.0 mg/l) and BA (0.5 mg/l) enhances the synthesis of total phenolic (9.20 mg GAE/g) and flavonoid (1.25 mg QE/g) content in the callus tissue. The callus produced by the same concentration shows 45.24 % of free radical scavenging activity. While comparing the various concentrations of NAA with 2,4-dichlorophenoxyacetic acid (2,4-D) for the production of callus biomass, total phenolic and flavonoid content and free radical scavenging activity, all the concentrations of NAA were found to be superior than those of 2,4-D.