In vitro shoot cultures of ragweed (Ambrosia artemisiifolia L.), an important weed and allergen species, were established and utilized to explore the phytotoxic effect of essential oils of Nepeta rtanjensis Diklic´ and Milojevic´, and N. cataria L. Ragweed shoots were exposed to the atmosphere enriched with volatile compounds emitted from essential oils which differ in their qualitative and quantitative nepetalactone content. Essential oil of N. rtanjensis, an endemic and critically endangered perennial in Serbia, was characterized by high amounts of 4aa,7a,7ab-nepetalactone (trans,cis-nepetalactone), while essential oil of N. cataria, possessed high amounts of 4aa,7a,7aa-nepetalactone (cis,trans-nepetalactone). After 2 weeks of exposure to Nepeta essential oil (2 and 4 %, final nepetalactone concentrations), in vitro morphogenesis of ragweed shoots was significantly altered. Reduction in fresh weight of shoots and roots and rooting inhibition was observed together with prominent discoloration of shoots. Alterations in antioxidative defense system of ragweed shoots as a response to essential oils treatments were characterized by increased peroxidase activity and decreased catalase and superoxide dismutase activity. Generally, essential oil of N. cataria, which possesses cis,transstereoisomer of nepetalactone, had stronger inhibitory effect on shoot growth, catalase activity, and was more efficient in stimulating peroxidase activity. N. rtanjensisessential oil, and thus trans,cis-nepetalactone, was more efficient in inhibiting rooting and root growth, and in suppressing superoxide dismutase activity. Therefore, essential oils of N. rtanjensis and N. cataria might find another application as potential bioherbicides against highly invasive species such as ragweed.