Fagonia indica is an important medicinal plant species used traditionally against a variety of diseases. In this study, we initiated callus cultures from healthy stem explants. We observed maximum callus induction frequency (88%) on MS media supplemented with Thidiazuron (1.0 mg/mL). We also examined the callus cultures to determine the impact of iron-doped zinc oxide nanoparticles (Fe-ZnO-NPs) in concentrations (15.62 to 250 µg/mL) on biomass accumulation, secondary metabolism, and antioxidative potential in callus cultures of F. indica. Our results showed that maximum callus biomass (FW = 13.6 g and DW = 0.58 ± 0.01) was produced on day 40 when the media was supplemented with 250 µg/mL Fe-ZnO-NPs. Similarly, maximum TPC (268.36 µg GAE/g of DW) was detected in 40 days old callus added with 125 µg/mL Fe-ZnO-NPs. Maximum TFC (78.56 µg QE/g of DW) was observed in 20 days old callus grown in 62.5 µg/mL Fe-ZnO-NPs containing media. Maximum total antioxidant capacity (390.74 µg AAE/g of DW) was observed in 40 days old callus with 125 µg/mL Fe-ZnO-NPs treated cultures, respectively. The antioxidant potential was in correlation with the TPC. These results showed that Fe-ZnO-NPs elicitors can increase the biomass and activate secondary metabolism in F. indica cells.