Purpose: To investigate the chemical composition, antioxidant properties and antibacterial effects of n-butanol extract of Lepidium sativum (Brassicaceae) seed. Methods: The antioxidant activity of extracts of the seed obtained by maceration was determined by 1,1-diphenyl-2 picrylhydrazyl (DPPH), reducing power, and 13-caroten/linoleic acid assays. The antimicrobial activity of the extract was tested against five strains of pathogenic microorganisms, viz, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, Enterococcus feacalis ATCC 29212, Escherichia coli ATCC 25992 and Pseudomonas aeruginosa ATCC 27852. Its phytochemical composition was determined by liquid chromatography-mass spectrometry (LC-MS). Results: A total of 17 compounds were identified by LC-MS, among which four compounds were kaempferol-hexose-rhamnose and three derivatives of kaempferol-rhamnose-hexose (benzoyl hexose) were previously unknown. The extract had better antioxidant than antibacterial properties. Conclusion: These results indicate that n-butanol extract of Lepidium sativum seeds possesses good antioxidant properties which are most likely due to the presence of quercetin and kaempferol. Thus, these compounds account for the use of Lepidium sativum in traditional medicine.