Trace minerals are essential components of fish that contribute to human nutrition and food security. In this study, Nile tilapia (Oreochromis niloticus) were collected from two restored basins of Lake Mariout, Egypt—the Main Basin (B1) and the Southwest Basin (B2)—to evaluate trace mineral accumulation, antioxidant enzyme activity, proximate composition, gene expression, and potential human health risks. Metal concentrations in fish tissues followed the order Fe>Ca>Zn>Cu, with Cu showing significant variation between basins. Human health risk assessment, including estimated daily intake (EDI), estimated weekly intake (EWI), and maximum daily intake (MDI), indicated no non-carcinogenic risk at consumption rates of 100–500 g per day. Proximate analysis showed that B1 fish had higher protein and carbohydrate content, whereas B2 fish exhibited significantly higher antioxidant enzyme activities. Gene expression analysis revealed upregulation in B2 fish of metalregulatory genes—metallothionein (mt1), divalent metal transporter 1 (dmt1), zinc transporter 1 (znt1), hepcidin 2 (hepc2)—and antioxidant defense genes—superoxide dismutase 1 (sod1), catalase (cat), glutathione peroxidase 1 (gpx1), glutathione reductase (gr), and glutamate-cysteine ligase catalytic subunit (gclc). Overall, Nile tilapia from the restored basins of Lake Mariout represent a nutritionally adequate and safe food source, highlighting the benefits of effective environmental management.