In the present study, some important pharmaceutical compounds in flaxseed plant have been done via biotechnology techniques. the seeds of Linum usitatissimum were surface sterilized germinated for one week on hormone-free MS culture media for 10 days. Cotyledonary leaves, hypocotyls segments (1 cm) and rootlets segments (1 cm) of the obtained sprouts were used as explants for callus induction on MS supplemented with BA (benzyladenine) and NAA (Naphthalene acetic acid). Maximum callus fresh weight was recorded in from rootlets explants and reached up to 3.72 gram in response to the treatment with 0.5 mg/l BA plus 0.25 mg/l NAA after 4 weeks of treatment. The highest callus fresh weight from cotyledonary leaves and hypocotyls segments were recorded in response to the treatment with 0.75 mg/l BA plus 0. 5 mg/l NAA and 0. 5 mg/l BA and NAA, respectively. The results obtained have revealed many differences between the resulted calli with respect to pharmacopeial constants and secondary metabolites determined (Phenolics, glycosides, flavonoids and saponins). Methanolic extracts of calli obtained from different explants exhibited antibacterial activity against both the Gram-positive and Gram-negative tested pathogenic bacterial strains. The maximum inhibition zone (26 mm) was obtained in response to the treatment of staphylococcus aureus with extract of callus obtained from cotyledonary leaf explants. This study may indicate that the callus of L. usitatissimum can be adopted in the future as a source of valuable metabolites.