Metalloenzymes play an important role in the regulation of many biological functions. An effective way to prevent deficiencies of essential minerals in human diets is the biofortification of plant materials. The process of enriching crop sprouts under hydroponic conditions is the easiest and cheapest to conduct and control. In this study, the sprouts of the wheat (Triticum aestivum L.) varieties Arkadia and Tonacja underwent biofortification with Fe, Zn, Mg, and Cr solutions in hydroponic media at four concentrations (0, 50, 100, and 200 µg g−1) over four and seven days. Moreover, this study is the first to combine sprout biofortification with UV-C (λ = 254 nm) radiation treatment for seed surface sterilization. The results showed that UV-C radiation was effective in suppressing seed germination contamination by microorganisms. The seed germination energy was slightly affected by UV-C radiation but remained at a high level (79–95%). The influence of this non-chemical sterilization process on seeds was tested in an innovative manner using a scanning electron microscope (SEM) and EXAKT thin-section cutting. The applied sterilization process reduced neither the growth and development of sprouts nor nutrient bioassimilation. In general, wheat sprouts easily accumulate Fe, Zn, Mg, and Cr during the applied growth period. A very strong correlation between the ion concentration in the media and microelement assimilation in the plant tissues (R2 > 0.9) was detected. The results of the quantitative ion assays performed with atomic absorption spectrometry (AAS) using the flame atomization method were correlated with the morphological evaluation of sprouts in order to determine the optimum concentration of individual elements in the hydroponic solution. The best conditions were indicated for 7-day cultivation in 100 µg g−1 of solutions with Fe (218% and 322% better nutrient accumulation in comparison to the control condition) and Zn (19 and 29 times richer in zinc concentration compared to the sprouts without supplementation). The maximum plant product biofortification with magnesium did not exceed 40% in intensity compared to the control sample. The best-developed sprouts were grown in the solution with 50 µg g−1 of Cr. In contrast, the concentration of 200 µg g−1 was clearly toxic to the wheat sprouts.