Antibiotics are crucial in the control of bacterial infections on fish farms. However, the influence of licensed antibiotics on DNA damage, histological changes, and protein differentiation in some farmed fish species is unidentified. The current study aimed to investigate the effect of therapeutic (80 mg/kg body weight/day) and high doses (160 mg/kg body weight/day) of oxytetracycline (OTC) on some biological indices, protein differentiation, histological changes, and DNA damage in Oreochromis niloticus (n = 360; 40 ± 3.5 g) and the potential positive effects of dietary ascorbic acid (AA) and whether it is capable of reducing the deleterious impacts of OTC and working synergistically to increase fish disease resistance and decrease morbidity and mortality rates. In the current study, fish were fed diets with or without OTC (80 or 160 mg/kg body weight/day) and AA (100 mg/kg diet/day) for 28 days, followed by a bacterial stress test with two different virulent strains for 14 days. Treatments include CTR (control; without additives), AA, OTC80, OTC160, OTC80 + AA, and OTC160 + AA. The results revealed that the AA group had the best antioxidative (significant increase in SOD, CAT, and GPx) and immunomodulatory effects (an increase in IgM, nitroblue tetrazolium, and total leucocytic count). Furthermore, the hematological parameters, immune indices (NBT), and antioxidant enzymes were significantly reduced by OTC160 (p ≤ 0.05). The results also showed that OTC160 notably augmented the activity of liver (ALT, AST, and LDH) and kidney (creatinine and urea) damage markers (p ≤ 0.05). OTC160 revealed considerable hepatic and renal tissue damage and significantly higher hepatocyte DNA damage. Pathological changes and DNA damage were directly correlated to the dosage of OTC exposure, where they were more severe in OTC160. OTC80 showed insignificant changes in most parameters. Compared to CTR, the inclusion of AA in the O. niloticus diet significantly restored the biological indices, histological changes, and DNA damage in OTC-treated groups. The morbidity and mortality percentages after bacterial challenge were significantly lower in the OTC80 + AA group, followed by the OTC160 + AA group than in the CTR. These results corroborate the use of AA as a potent antioxidant and immune stimulant and a potential co-adjuvant to OTC in the diet of O. niloticus to reach optimal resistance to disease.