Using immunostimulants and acidifiers and their salts as antibiotic alternatives and growth promoters has become important due to the emerging antimicrobial resistance and is currently employed in livestock and aquaculture feed. So, this study is designed to assess the ameliorative properties of sodium butyrate (SB) on Oreochromis niloticus growth indices, immune responses, antioxidant, growth-related gene expression, and its resistance against the Pseudomonas aeruginosa infection. Five groups of O. niloticus were distributed (30 fish/group); the first control (CTR) group was fed a basal diet without any additives, while the 2^nd to 5^th groups were fed on a SB supplemented diet with 0.25, 0.50, 0.75, and 1% concentrations, respectively. After 8 weeks, all groups were subdivided into; the diet-treated group with enrofloxacin and the untreated ones. Following the consumption of the medicated feed, all groups were infected with P. aeruginosa. The SB-fed fish showed an upregulated expression of growth-related genes (GH and IGF-1) and the antioxidant enzyme genes (catalase, and superoxide dismutase). The hematological parameters, immune indices, bactericidal activity, and phagocytosis were improved. After P. aeruginosa infection, a high mortality rate was recorded in the CTR-infected untreated group (50%), while it was markedly reduced (10%) in 0.75% and 1% SB-infected untreated groups. The enhanced potential synergistic effect of SB in 0.75% and 1% with ENRO was also observed. Therefore, diet supplementation with sodium butyrate, particularly at 0.75%, either alone as a growth enhancer or combined with ENRO, is recommended to protect fish completely from P. aeruginosa infection.