The analysis of the extract of Lepidium sativum seeds revealed the presence of some chemical groups (total polyphenols and flavonoids) likely to express beneficial activities on health. Therefore, the extract was screened for its antimicrobial activities against several pathogenic strains and antioxidant activities in vitro. The extract reacted positively on the tested microbial strains and showed that it can act as a free radical scavenger. In addition, its anti-inflammatory and analgesic activities were explored. The exploration of the anti-inflammatory activity in vitro showed that the extract has a strong capacity to inhibit protein denaturation up to 70.5%. In vivo, the extract has proven to be significantly effective in reducing carrageenan-induced plantar edema by 80.26%, and it has also demonstrated significant analgesic activity in reducing acetic acid-induced contortions by 40%. Also, the administration of the extract exerted an inhibition of 72.94% of the vascular permeability induced by acetic acid.