Brassica juncea L. (Brassicaceae) is widely used in making mustard oil. The petroleum ether extract of the seeds of this plant found to contain appreciable levels of secondary metabolites including phenolics, flavonoids, tannins and alkaloids that might be attributed to the good antioxidant activity of this extract. The extract exhibited broad antimicrobial activity against the tested pathogenic strains including Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumonia, Candida albicans, Escherichia coli and Proteus vulgaris. 10 compounds were identified in the petroleum ether extract of Brassica juncea (L.) seeds using gas chromatography- electron ionization mass spectrometry (GC-EIMS) analysis. The identified constituents were  9,19-Cyclolanost-24-en-3-ol, acetate, (3.beta.)- Cycloartenyl acetate (34.77%), 3-Methylpentane (25.26%), Methyl cyclopentane (15.44%), octadec-9-enoic acid (oleic acid) (9.02%), Allyl isothiocyanate (3.43%), 3-Butenyl isothiocyanate (3.38%), hexanal (Caproaldehyde) (3.14%) and nonacosane (0.95%). In conclusion, Brassica juncea seeds could be used as valuable source of active constituents that possess remarkable biological activity and could be used in the field of medicine.