Stevia (Stevia rebaudiana Bertoni) is an essential herbal plant used as a sweetener. The demand for stevia is growing due to its low caloric and medicinal value, hence the need for a more thorough investigation of its nutritional and biological properties. Nanoparticles of metal oxides have been found to have broad applica-tions in agriculture for the stimulation of plant growth and development. The study aimed to assess the effect of various zinc oxide nanoparticles (ZnONPs) concentrations on stevia plants’ quantitative and qualitative traits obtained in in vitro cultures. Micropropagation of two stevia varieties, Candy and Morita, was carried out using explants of shoot tips placed on MS medium supplemented with 1.0 mg dm–3 BA and 0.1 mg dm–3IBA and with ZnONPs at concentrations of 0 (control), 10, 20, 30 and 40 mg dm–3. The obtained results indicated that high concentrations of ZnONPs stimulated the propagation of shoots. On the other hand, they negatively influenced shoot length, root number and length, and the fresh weight of the plantlets. The presence of zinc oxide nanoparticles in the medium increased the potassium, calcium, magnesium, and zinc content while decreasing the sodium and iron content in the regenerated stevia plantlets. The total phenolic content in the Candy variety was higher in the treatments with ZnONPs than in the control plants, while it was varied in the Morita variety.In both varieties, total antioxidant content measured by the ABTS method showed significantly higher in the treatments with 20–30 mg dm–3 ZnONPs than in the control. The content of chlorophyll a, chlorophyll b and chlorophyll a + b in the Morita variety was higher in the treatments with 10 and 20 mg dm–3 ZnONPs than in the control. On the other hand, high concentrations of ZnONPs negatively affected the content of carotenoids in both varieties. The study showed that stevia plants obtained in in vitro cultures on control media and media containing ZnONPs had a high content of valuable minerals, phytocompounds with antioxidant properties, and photosynthetic pigments.