Background: Lepidium vesicarium (Cruciferae), one of the important medicinal plants with a long history of medicinal use. The current study was designed to evaluate the free radical scavenging and antimicrobial activities of the L. vesicarium EO as one of the Iranian plant species. Methods: The compositions of the EO from the aerial parts of L. vesicarium were analyzed by GC-MS and GC-FID. Furthermore, anti-oxidant and anti-microbial potentials were investigated via DPPH reagent and disk diffusion procedure, respectively. Results: A total of 18 compounds amounting 97.70% of the oil have been identified, while Benzyl cyanide (43.94%), Isothio cyanic acid (22.69%) and Benzyl isothio cyanate (20.69%) were the main constituents. The EO showed no activity against the free radicals and studied microbial strains (gram positive and gram negative and also fungi species). Conclusion: On the whole, the presence of cyanide derivatives in studied EO revealed the rational use of this plant in medicine. Comparing with other genesis of Lepidium, anti-oxidant and anti -microbial properties of L. vesicarium essential oil were not noticeable.