This study aimed to conclude the impacts of three different dosages of formic acid (an organic acid) on various biological and physiological parameters in the mono-sex Nile tilapia (Oreochromis niloticus). The study examined growth performance parameters, hematological and serum biochemical profiles, and the activity of antioxidant enzymes. One hundred and eighty healthy fish, weighing about 30±5g, were stocked in glass tanks (60L/volume) to acclimate. There were 45 fish in each group (G1, G2, G3, and G4). Over eight weeks, groups 2, 3, and 4 were given experimental diets containing formic acid at concentrations of 0.3%, 0.4%, and 0.5%, respectively. Group 1 was used as a control and kept on the basal diet. Groups given diets supplemented with 0.4 and 0.5% formic acid demonstrated a noticeably better feed utilization and weight gain than the 0.3% supplementation group. Overall, during the first 4 weeks, FCR was significantly enhanced compared with the following 4 weeks. The numbers of red blood cells (RBCs), platelets, hemoglobin, white blood cells (WBCs), and serum proteins (total protein, albumin, and globulin) were almost unchanged across all groups. Even liver function and creatinine levels were unchanged. On the other hand, formic acid at the concentrations of 0.4 and 0.5% showed significant improvements in antioxidant enzyme activity (SOD, CAT, and TAC), while the 0.3% group showed a moderate decrease compared to the control. Additionally, when fish was challenged by Aeromonas hydrophila, the 0.5% formic acid group had the lowest mortality rate (MR%). The 0.3% group showed better immunological responses, with increased serum bactericidal activity (SBA) and oxidative burst activity (OBA). These results point to formic acid as a potential feed addition for improving growth, boosting immunity, and attaining sustainable aquaculture methods. Dietary formic acid is not recommended to exceed the 4 weeks addition, high period may cause a decrease in FCR.