Probiotics play a significant role in aquaculture by improving the growth, health, and survival rate of fish against pathogenic organisms. In the present study, we have evaluated the effects of aLactobacillus rhamnosus(L. rhamnosus) probiotic on growth performance and disease resistance inOreochromis niloticus(O. niloticus) fingerlings. Four different concentrations ofL. rhamnosus(T1: 0.5 × 1010, T2: 1 × 1010, T3: 1.5 × 1010, and T4: 2 × 1010CFU/kg feed) were administered to fish over a period of three months.L. rhamnosustreated fish revealed a high growth increment as compared to the control, and the values of macromolecules (amino acids, fatty acids, and carbohydrates) varied significantly among the treated and control groups. Levels of thyroid hormones were noted to be high in the probiotic-treated groups. A challenge assay was performed withAeromonas hydrophila(A. hydrophila). The optimum calculated concentration of probiotics from the growth assay (1.5 × 1010CFU/kg feed) was used for the challenge assay. Fish were divided into four groups as follows: control (Con), probiotic-treated (PL), infected (I), and infected + probiotic-treated (I + PL) groups. Significant variations in hematological parameters were observed among control and treated groups. Histopathological changes were recorded in infected fish, while the infected + probiotic-treated group showed less deformations indicating the positive effect of the probiotic supplementation. The survival rate of fish was also better in the probiotic-treated group. Based on these findings, we conclude that probiotic supplementation enhances the growth and improves immunity ofO. niloticus. Therefore, we propose that probiotics can be used as promising feed supplements for promoting fish production and disease resistance in aquaculture.