Oreochromis niloticus is imperative in Indian aquaculture attributing to a significant portion of culture fisheries after carps. This study assessed the serum biochemical, haematological, and histopathological changes in O. niloticus juveniles upon dietary administration of florfenicol (FFC) at 15 mg/kg biomass/day (1X) and 45 mg/kg biomass/day (3X) for 10 consecutive days and compared with control. Behavioural changes, feed consumption, survival, and biomass were evaluated. Besides, the levels of serum glucose, calcium, chloride, creatinine, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, and blood cell morphology were determined at scheduled intervals. The 10 days of oral dosing resulted in 100% (1X) and 96.15% (3X) survival, reduced feed intake, and biomass. Significant erythrocyte morphological alterations were observed in the 1X group compared to the control. Alterations in haematological parameters of FFC-dosed fish suggested apparent cytotoxicity. A substantial hike in lymphocyte counts and mean corpuscular volume was observed. All the serum biomarkers of O. niloticus increased significantly on day 10 dosing in a dose-dependent manner, except for calcium and chloride, which reduced significantly. Marked histopathological changes like degeneration of renal tubular epithelium and widened lumen in the kidney, and glycogen-type vacuolation, cytoplasmic degeneration, and cytoplasmic vacuolation in the liver were observed during the dosing period. Within 3 weeks of suspension of dosing, the 1X group serum biomarker levels became normal except for alkaline phosphatase. The recovery of serum biomarker levels, haematological values, histopathological and erythrocyte morphological changes suggested that the FFC-induced changes are reversible at the therapeutic dose and are safe for O. niloticus.