Pseudomonas putida is a virulent bacterium that prompts major losses in fish. Recently, there has been a noticeable direction for utilizing nanomaterials in the aquaculture industry for sustaining fish health and per formance. Hence, the present study is the first trial to investigate the antibacterial influence of nano titanium dioxide gel (NTG) as a watery addition for combating P. putida infection in Nile tilapia (Oreochromis niloticus). Further, antioxidant-immune capacity, and gene expression in the spleen including antimicrobial peptides and immune-related genes are assessed. Fish (n = 200; 47.50 ± 1.32 g of body weight) were assigned into four groups for 10 days [control, NTG (0.9 mg/L), P. putida, and NTG + P. putida]. Findings demonstrated that the infection by P. putida induced a decline in antioxidant immune indicators including catalase, glutathione peroxidase, and nitric oxide. Furthermore, a noteworthy rise in lipid peroxide (malondialdehyde), tumor necrosis factor-alpha (TNF α ), and stress indicator (glucose) levels was noticed. P. putida infection induced remarkable alterations in the expression of antimicrobial peptides genes [tilapia piscidin (TP3 and TP4), colony-stimulating factor 1 re ceptor, hepcidin-2, beta-defensin1, and neutrophil cytosolic factor 4] and immune-relevant genes [transforming growth factor beta, tumor necrosis factor receptor-associated factor 6, TNF α , interleukins (IL-10 and IL-11)]. Notably, applying NTG regenerated all the negative consequences of P. putida infection. Inclusive, this study underscores the crucial role of NTG as a potent antibacterial and immune-antioxidant agent, highlighting its potential in protecting O. niloticus from P. putida infection and improving immune-antioxidant response.