The essential oil obtained from Dysphania ambrosioides, a fragrant plant growing wildly in Tunisia, was extracted by hydrodistillation and analyzed using GC-MS and GC-FID techniques. Forty-four components were identified in the essential oil (EO), representing 88.95 % of the total hydrodistilled oil. The most abundant components were cis-ascaridole (60.33 %), m-cymene (22.17 %) and α-terpinene (1.79 %). The oil obtained from Tunisian D. ambrosioides, showed a notable antiviral activity against CV-B4 virus with IC50 equals to 21.75 μg/mL. The EO showed no toxicity on the HEp-2 cell line as evidenced by its low Cytotoxic Concentration 50 (CC50) that is estimated by 1617.22 μg/mL with a good selectivity index (SI = 74.348). D. ambrosioides essential oil showed a notable antiviral activity against CV-B4 virus with IC50 equals to 21.75 μg/mL. The EO showed strong antibacterial activity against Pseudomonas aeruginosa and Bacillus subtilis bacterial strains as well as antifungal activity against the pathogenic strain Candida albicans. The encouraging results indicate that D. ambrosioides essential oil might be explored as a natural source of antiseptic products relatively safer comparable to versatile synthetic alternatives. Meanwhile these results should be further supported by clinical trials to confirm the claimed activity.