Development of alternative disease treatment and prevention strategies has received considerable attention in recent years due to frequent epizootic outbreaks in modern intensive aquaculture. Poly-β-hydroxybutyrate (PHB) is a biopolymer synthesized by some bacteria with potential immunostimulatory effects. This study analyzed the potential of PHB as an immunomodulator in juvenile Nile tilapia utilizing in vitro and in vivo approaches. Head-kidney-derived macrophage production of intra- and extra-cellular superoxide anion exhibited significant linear and quadratic relationships with graded doses (0.0, 0.5, 1.0, 2.0, 4.0 and 8.0 mM) of a PHB precursor, 3-hydroxybutryate (3HB). Additionally, lyophilized PHB-synthesizing bacteria, Zobellella denitrificans (ZD1), were supplemented to a practical basal diet for Nile tilapia (36% crude protein and 6% crude lipid) to produce five isonitrogenous and isolipidic experimental diets containing PHB in five stepwise increments (0.125, 0.25, 0.5, 1.0, and 2.0% of dry-diet weight). After termination of the feeding trial, Nile tilapia exhibited dose-dependent linear and quadratic relationships in percentage weight gain, as well as feed efficiency, protein conversion efficiency, and hepatosomatic index. Finally, PHB-supplemented diets did significantly modulate intestinal microbiota composition. In conclusion, Nile tilapia exhibited enhanced growth parameters, condition factors, immunological responses, and intestinal microbiota modulation when fed PHB, with the optimal inclusion level determined to be 1% of dry diet.