In this study, the impact of Fe3O4 nanoparticles (NPs) at different concentrations was studied on physiology, antioxidant enzymes, cell damage, and secondary metabolites of Physalis alkekengi seedlings under in vitro conditions. Seeds were grown on a solid MS medium containing different Fe3O4 NPs concentrations (0, 10, 20, and 30 mg/L). The results showed that Fe3O4 NP at 20 mg/L increased growth (fresh and dry weight, length of shoot and root, and the number of adventitious roots) and physiological (relative water content, chlorophyll pigments, carotenoid, carbohydrate, H2O2, and ion leakage) parameters through stimulation of protein content and antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase. Fe3O4 NP at 20 mg/L was found to be the optimum concentration for enhancing secondary metabolites (total flavonoids, phenolics, and withanolides) and activity of DPPH radical scavenging and PAL, while the higher Fe3O4 NPs concentration (30 mg/L) showed toxicity impact on growth parameters through promoting H2O2 level, lipoxygenase activity, and ion leakage and inactivation of the antioxidant enzyme isoforms. The electrophoretic pattern displayed Fe3O4 NPs at 30 mg/L decreased the band intensity of SOD (Mn-SOD and Cu/Zn-SOD1) and CAT (CAT1 and 2) isoforms. These findings display that the optimal dose of Fe3O4 NPs (20 mg/L) acts as an elicitor to induce antioxidant functions of P. alkekengi cell system to suppress oxidative damage.