Bio-fortification is a potential technique to tackle micronutrient deficiencies that remain. Wheat grain bio-fortification has the ability to decrease malnutrition because it represents one of the most essential staple crops. Bio-fortification is cost-effective and evidence-based sustainable technique to address malnutrition in wheat varieties possessing additional micronutrient contents. Nano-biofortification is a novel approach, enriching crops with essential nutrients in order to supplement human diets with balanced diets. The current study was designed to explore the potential role of phytogenic iron nanoparticles (Fe-NPs) to enhance nutritional contents in wheat plants to fulfill the nutrient deficiency important for human and animal health. In the current study, Fe-NPs were fabricated by using the extract of Mentha arvensis L. that were irregular in shape with an approximate size range of 40–100 nm. Further, Fourier transform infrared (FT-IR) analyses were deployed to confirm the presence of t of various functional groups involved in the green and eco-friendly fabrication of Fe-NPs. The effects of phytogenic Fe-NPs were examined on various physiological and biochemical parameters such as total proline, total chlorophyll, carbohydrates, protein, crude fibers, and lipids contents. Moreover, wheat physiological and biochemical profiling was carried out, and it was noticed that Fe-NPs significantly altered the physico-biochemical profiling of wheat plants. Multiple methods of administration of Fe-NPs were used to fortify the wheat crop. However, the Fe-NPs assisted seed priming along with foliar applications at various concentrations (10, 20, and 30 mg·L −1 ) were found more suitable to enhance the contents of proline, Chlorophyll a, b, total chlorophyll, carbohydrate, proteins, fibers, and lipids (20.22%, 18.23%, 17.25%, 16.32%, 12.34%, 24.31%, 19.52%, and 11.97%, respectively) in wheat plants. Further, wheat flour was exposed to digestive enzymes, with the iron content gradually increased in a dose-dependent manner. The nutritional analysis of wheat zinc (Zn), molybdenum (Mo), magnesium (Mg), iron (Fe), yttrium (Y), and copper (Cu) and the fatty acid profile have demonstrated divergent patterns of behavior. Similarly, iron content was also increased significantly in response to the exposure to Fe-NPs.