The immune system of fish is severely impacted by heavy metal pollutants that enter the aquatic system, reducing their survival and resistance to diseases. Arsenic is a widespread heavy metal that contaminates aquatic bodies, impacts fish health, and hinders the sustainability of aquaculture. In the current investigation, we studied the arsenic-hazard consequences on the hematology, serum immunological responses, antioxidant activity, autophagy-related genes, splenic architecture, and protection against Aeromonas sobria in Nile tilapia (Oreochromis niloticus). Additionally, we proposed the ameliorative potential of genistein as a feed additive against arsenic exposure. For this purpose, 160 fish were equally set into four groups, each with four replicates for sixty days. The C (control) and GN (genistein) groups were fed basal diets fortified with 0 and 500 mg genistein/kg diet and were not exposed to arsenic. However, the AS (arsenic) and AS + GN (arsenic + genistein) groups were reared under arsenic conditions (10 µg/L of As2O3) and fed on identical diets as the C and GN groups. The outcomes revealed that arsenic exposure induced hematological disorders and anemic conditions in Nile tilapia. The serum immunological and splenic antioxidant enzymes (phagocytic activity, nitric oxide, lysozymes, complement 3, superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione S-reductase, and reduced glutathione) were substantially lowered, and the malondialdehyde level was increased in the arsenic-exposed fish. Arsenic exposure down-regulated the splenic expression of antioxidant enzymes while upregulated the autophagy-related genes (Beclin-1 and AKT-1) and endoplasmic reticulum (ER) stress-related genes (ATF-6, XBP-1, JNK, and BIP). The arsenic-exposed fish showed the lowest survivability (66.7%) when challenged with A. sobria relative to the control (80%). The dietary intervention of genistein in the Nile tilapia diet mitigated the arsenic-induced hematotoxic, immunotoxic, oxidative stress, autophagy, and ER stress consequences. The histological picture of the spleen and the survivability of the arsenic-exposed fish were restored by feeding on the genistein-fortified diet. Genistein is a promising feed additive for counteracting the harmful effects of arsenic in Nile tilapia, which helps in sustainable aquaculture development.