Aquaculture has been facing numerous obstacles, particularly diseases causing great financial loss. A twelve-week trial was conducted to assess a strategy of intermittent dietary of mannan oligosaccharides and β-glucan (active-MOS) on the immunity of the Nile tilapia. Five groups (G1–5) were fed on a supplemented diet for 12 weeks, interrupted with a one-week-free diet every 0, 1, 2, 3, and 12 weeks. Growth parameters FW, WG, DWG, RGR, and FCR were significantly enhanced after supplementing of dietary-MOS compared to the control group, with a superiority of G3 122g, 70.7g, 0.83g, 134.95 and 1.57, respectively. The activity of serum antibacterial, oxidative burst, and phagocytosis (innate immunity) was significantly boosted in supplemented fish regardless of the supplementation period. The gene expression of interleukin (il)-1β, Heat shock protein (Hsp)-70, and tumor necrosis factor (tnf)-α were increased in response to dietary-active-MOS supplementation. They were significantly higher in G3 and G4 fish, recording values of 4.87, 0.81, and 7.1; 3.7, 0.86, and 6.74, respectively, compared to the control group 0.68, 0.15, and 2.3 fold change, respectively.  Dietary-active-MOS induced the gene expression of catalase and superoxide dismutase (antioxidant enzymes); fish of G4 had significantly high gene expression with 0.7- and 2.89-fold changes, respectively. On the challenge test with Aeromonas hydrophila, the Nile tilapia fed dietary-active-MOS had a low mortality rate (35%), providing a high relative level of protection (41.67%) in G3 and G4. Thus, dietary-active-MOS is recommended with a week interval of free additive diet to boost the immunity of the Nile tilapia and provide protection against bacterial infection.