Chemical fertilizers used in plant development and differentiation have become a global problem affecting the entire ecosystem, especially soil pollution. Food production demand with the increasing population has encouraged scientists to use biogenic nanoparticles in the agricultural field. Evaluation of growth, development, and differentiation processes of sweet basil (Ocimum basilicum L.) seedlings at gradually increasing concentrations of biogenic iron oxide nanoparticles (BIO-NPs) were identified by morphological and physiological parameters in this study. The results showed that growth parameters reached the maximum value at 100 mg/L but were less at other concentrations. At similar concentration, the stomatal density of the leaf was the maximum, while the stomatal area showed the lowest value. The levels of H2O2 and malondialdehyde (MDA) decreased in the treated seedlings. BIO-NPs increased the antioxidant defense and supported its growth by changing the antioxidant enzyme activities, H2O2, and MDA contents. The BIO-NP treatment provided positive improvements in phytochemical content in parallel with the growth and development of sweet basil seedlings. Different growth parameters, physiological results, supporting enzyme activities, and biochemical data revealed the contribution of the BIO-NP treatments to the growth and development of sweet basil seedlings. BIO-NPs improved higher phytochemical production of sweet basil, which may be suitable for its propagation on a commercial scale.