The development of knowledge on bread wheat (Triticum aestivum L.) biofortification in zinc (Zn) and iron (Fe), related to its potential agronomical use and the nutritional and technological implications, is becoming important to strategies for improving human nutrition. In this context, we studied the accumulation of Zn and Fe in grains, considering potential uptake and translocation kinetics, photoassimilate production and deposition, and related yields, in grains of cv. Roxo produced under controlled-environment conditions and used thereafter in field trials. The metabolic plasticity of this wheat genotype grown under controlled-environment conditions allowed a 10- and 4-fold enhancement in accumulation of Zn and Fe in the grains after nutrient supplementation with a 5-fold concentrated Hoagland solution (5S), after two generations. Moreover, when these seeds were sown under field conditions and the resulting plants supplemented with or without Zn and Fe, the accumulation of these nutrients decreased within the next two generations. Such field seeds obtained without further Zn and Fe supplementation (with nitrogen only; F_3(S) and F_4(S)) maintained enhanced levels of Zn (∼400%) and Fe (40–50%) compared with the initial seeds. If Zn and Fe supplement was given to the plants germinated from F_2(5S), the subsequent F_3(5S) and F_4(5S) seeds maintained the Zn increase (∼400%), whereas a further enhancement was observed for Fe, to 75% and 89%, respectively. Toxic limits were not reached for photosynthetic functioning. Even under the highest Zn and Fe supplement dose given to the F_3(5S) plants, there was only a slight effect on photosystem II photochemical performance; in fact, enhanced net photosynthesis values were observed. In conclusion, within this experimental design, Zn and Fe biofortification can be obtained without toxicity effects on photosynthetic performance and with negligible modifications to grain texture and nutritional value (protein quality and contents as well as fatty acids).