Water temperature significantly influences fish behavior and physiology. Thus, the current study assessed the effects of dietary incorporation of Spirulina-co-enzyme Q10 nanoemulsion (SCN) on various health parameters in Nile tilapia (Oreochromis niloticus) raised in heat stress conditions (32 °C) for 60 days. In a completely randomized design, Nile tilapia (n=225) were divided into 5 groups (3 replicates, 15 fish per group). The first (C25) and second (SCN0) groups were fed a basal diet and reared at 25 °C and 32 °C, respectively. Groups 3–5 were reared at 32 °C and fed a diet supplemented with SCN at 10 mg/kg (SCN10), 20 mg/kg (SCN20), and 40 mg/kg (SCN40), respectively. The findings indicated that the inclusion of SCN in the diets of O. niloticus, particularly at 20 and 40 mg/ kg concentrations, significantly (P<0.05) counteracted the heat stress (32 °C) impact on behavior, hematological parameters, stress biochemical indicators, additionally stress related genes and histopathological changes in fish, were assessed. SCN0 expressed decline in feeding frequency and mid-water swimming behavior, surfacing and high opercular movement. Cholesterol, triglycerides, glucose, and cortisol levels were significantly decresead with SCN supplementation. Heat stress induced significant reduction (p<0.05) in the hematological indices (RBCs, WBCs, Hb, and HCT%), which were ameriolated after SCN addition. SCN also significantly decreased encephalopathic alterations (vascular congestion, perivascular edema, neuronal pyknosis associated with perineuronal vacuolation, and neuropil microcavitation) in the brain of fish treated at 32 °C. The incorporation of SCN into the diet markedly prevented the abnormal expression of hsp70, tgf-β, p53, Chop, and Bip in the brain of heat-stressed fish. In conclusion, SCN at rate of 20 mg/ kg is a promising dietary supplement capable of mitigating the negative effects of heat stress in Nile tilapia.