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The <i>Pseudomonas putida</i> strain was primarily identified and tested in vitro against antibiotic sensitivity for several antibiotics using the disc diffusion method. This isolate was also tested against sensitivity to carvacrol oil (c) and formic acid (f). The genotyping of <i>Pseudomonas</i> spp. and virulotyping for <i>P. putida</i> isolate was carried out and verified by 16S rDNA-PCR amplification. Furthermore, we assessed the efficacy of carvacrol oil and formic acid in vivo for treatment of <i>P. Putida</i> infection. For the in vivo challenge, 180 fish (<i>Nile tilapia</i>, <i>Oreochromis niloticus</i>) were divided into six groups: (G1: control (unchallenged), G2: carvacrol prophylaxis (3 g/kg), G3: formic acid prophylaxis (5 mL/kg), G4: control positive (challenged), G5: carvacrol treatment (3 g/kg), and G6: formic acid treatment (5 mL/kg); 30 fish per group) with three replicates. Following the challenge, nitric oxide and lysozyme activity were measured as essential indicators for fish immunity. The antioxidant parameters including SOD and catalase were computed to reflect the antioxidant status. Furthermore, relative percent survival (RPS) and mortality percent were evaluated to indicate functional immunity. The findings of the antibiotic sensitivity test showed that ciprofloxacin exhibited the largest inhibition zone. Additionally, formic acid (f) displayed the greatest inhibition zone compared to carvacrol oil (c) and was more effective in stimulating the immune-antioxidant response compared to carvacrol oil. The tested exotoxin A (tox A), exoenzyme S (exo S), and the nan1 associated-virulence genes were identified in the <i>P. putida</i> isolate. Overall, the current study verified the virulence of <i>P. putida</i> and highlighted the promising role of dietary addition of formic acid for enhancing the immune-antioxidant indicators and for mitigating <i>P. putida</i> infection. Future studies could be devoted to this field.