Aeromonas hydrophila is a major pathogen associated with serious production losses in the culture of Oreochromis niloticus. The present study was conducted to find potential probiotics from saline tilapia green water that could prevent A. hydrophila infection in juvenile tilapia. Several presumptive bacterial probiotics were isolated from saline tilapia green water and tested for inhibitory activities against A. hydrophila.  Bacterial isolate exhibiting inhibitory activity against A. hydrophila was further tested for pathogenicity against the host fish. Gut colonization activity and optimum probiotic dose to inhibit A. hydrophila colonization in tilapia gut were conducted. The infection challenge test was addressed to evaluate the protective effects of the isolated probiotics against A. hydrophila infection in juvenile tilapia. Results showed that among the presumptive probiotic isolates, Staphylococcus aureus showed the highest zone of inhibition and was used in the experiments. No mortalities or disease signs were found in juvenile tilapia after a 30-day challenge test with the isolated S. aureus. Dietary supplementation with a probiotic dose of 10⁶ CFU/ml was found optimal for gut colonization and inhibition of A. hydrophila colonization. The infection challenge test indicates that fish receiving 10⁶ CFU/ml S. aureus supplemented diets were protected against pathogenic A. hydrophila infection. Collectively, the application of 10⁶ CFU/ml S. aureus as a probiotic is a practical approach to prevent A. hydrophila infection in tilapia aquaculture.