The present study was carried out to develop an efficient plant regeneration protocol for Sesamum indicum (L.) from cotyledon explants prepared from 10-day-old in vitro grown plantlets. Cotyledon explants inoculated on MS medium containing 2 mg l⁻¹ N⁶ –Benzyladenine (BA) and 30 mg l⁻¹ spermidine induced the higher number of shoots (23.43 shoots/explant) and the induced shoots were elongated (5.63 cm/shoots) in the same medium. The maximum frequency (92.33%) of root induction was recorded in MS medium fortified with 10 mg l⁻¹ putrescine. Up to 95% of the regenerated plantlets acclimatized and developed further under the greenhouse conditions. Antibacterial activity of the regenerated plant extracts exhibited resistance against various pathogens such as, Escherichia coli, Vibrio cholerae, Bacillus subtilis and Micrococcus luteus. Highest zone of inhibition (13 mm) was recorded for B. subtilis and M. luteus at 50 μg ml⁻¹ extract.