This study aimed to investigate the effects of dielectric barrier discharge cold atmospheric plasma on the performance of basil (Ocimum basilicum L. cv. Genovese Gigante). Evaluations were carried out on several physiological and biochemical traits, including ion leakage, water relative content, proline and protein accumulation, chlorophyll and carotenoid contents, and antioxidant activity. Before planting, basil seeds were treated by cold atmospheric plasma under voltages of 10, 15, and 20 kV for 10, 20, and 30 min. The ion leakage rate in plants was significantly affected by the interaction between plasma and radiation time. In most treatments, the application of plasma significantly reduced the ion leakage rate. The application of plasma (10 and 20 kV) for 10 min significantly increased the relative water content of basil leaves. The maximum amount of total chlorophyll and carotenoid content occurred after applying plasma for 20 min with 15 kV. Furthermore, 10 and 15 kV treatments of atmospheric cold plasma for 10 min caused a significant increase in antioxidant activity. The highest total flavonoids were obtained after applying 15 kV treatments for 20 min and 20 kV for 30 min, respectively. Cold atmospheric plasma significantly increased the activity of peroxidase as an antioxidant enzyme. Moreover, the minimum and maximum values of microbial load based on logarithm ten were reached after applying 10 kV for 30 min and in the control group, respectively. In general, the results showed that dielectric barrier discharge cold atmospheric plasma could significantly improve basil plants’ physiological and biochemical traits.