Abstract <p>The presented research is devoted to a potential of MgO nanoparticles of size 4–8 nm in various concentrations (25, 50, 75, 100, 150, 200 mg/L) application for enhancement of red-leaved sweet basil (<i>Ocimum basilicum</i> L.) of the ‘Khaki’ variety growth and essential oils accumulation in plants in total control environment agriculture. Concentrations from 25 to 75 mg/L had a stimulating effect on the growth and development of basil plants: the height increased by 8–17%, maximum achieved increase in the number of leaves (51%) and the dry weight (16.9%) with use of 75 mg/L compared to control. At the same time, concentrations of 25–100 mg/L of MgO-NPs did not affect the of essential oil content (on average, 0.13 µL/g). An increase in concentrations till 200 mg/L caused a stress reaction in plants (increase in superoxide anion—35 times and in hydrogen peroxide—1.6 times), which led to an increase in the essential oil content, reaching 0.21 µL/g at treatment with 200 mg/L (80% increase relatively to the control). However, high dosage of nanoparticles (150 and 200 mg/L) had an inhibitory effect on plant growth, reducing height by 15–36%, reducing the small leaves area and quantity, while not significantly affecting the fresh and dry weight. Thus, the use of magnesium oxide nanoparticles at a concentration of 200 mg/L can significantly increase the essential oil yield, despite the accompanying stress on the plant.</p>