Bacterial infection is one of the significant problems in fish farming that causes financial losses. With their anti-bacterial potential, metal-based nanoparticles are an innovative and successful disease control and prevention approach. It is hypothesized that zinc oxide nanoparticles synthesized by Nelumbo nucifera leaves (NN-ZnONPs) may be able to lessen deleterious effects induced by Clostridium perfringes type A challenge in Oreochromis niloticus (Nile tilapia). In vitro, NN-ZnONPs exhibited anti-bacterial efficacy against C. perfringes with a 22 mm inhibitory zone. A first experiment with 90 fish (average body weight = 40 ± 1.23 g) was conducted to identify the safe, appropriate amount of NN-ZnONPs, and it was found to be 1.5 mg/L. The fish (n = 160) were randomized into four groups for ten days, with the first group (control) and the second group (NN-ZnONPs) receiving 0 and 1.5 mg/L NN-ZnONPs as a water additive, respectively. The third (C. perfringes) and fourth (NN-ZnONPs + C. perfringes) groups were receiving 0 and 1.5 mg/L NN-ZnONPs, respectively, and challenged with C. perfringes (1 × 108 CFU/mL). C. perfringes infection caused the highest mortality (47.5%) with a marked increase in hepato-renal, stress (cortisol and glucose) indicators, and lipid peroxidation (malondialdehyde, MDA). On the contrary, significant declines in the protein profile (total proteins, albumin, and total globulins), antioxidants (total antioxidant capacity, catalase, and reduced glutathione content), and immune response (nitric oxide, total immunoglobulin M, lysozyme activity, and complement 3) were observed in the C. perfringes group. Remarkably, the NN-ZnONPs + C. perfringes group showed an improvement in these metrics compared to the challenged-non-treated group (C. perfringes). These findings indicate that NN-ZnONPs (1.5 mg/L) are protective against the detrimental effects of C. perfringes type A infection in aquaculture.