This study investigated the potential of biogenic ZnO nanoparticles (ZnO-NPs) to alleviate cadmium (Cd) toxicity in durum wheat plants exposed for 14 days to 25 μM CdSO4. By applying ZnO-NPs at two different concentrations (25 and 50 mg L−1), we observed increased chlorophyll content, beneficially impacting the photosynthetic efficiency, and enhanced sulfur, zinc, and iron accumulation. Moreover, the ZnO-NP treatment reduced the Cd accumulation in shoots, mitigating leaf chlorosis and oxidative damage. This response was clearly mediated by the increased thiol and phytochelatin production, as well as the enhanced sulfate uptake rate, with TdSultr1.3 as the most responsive gene coding for high-affinity transporter to Cd stress. In conclusion, the application of biogenic ZnO-NPs appears to be a promising approach for reducing the uptake of heavy metals by plants. In addition, it could be successfully used in combination with contamination prevention measures and/or remediation of contaminated sites to remove and mitigate the harmful effects of Cd on the environment and human health.