The pharmacokinetic of a drug describes a changes concentration of a drug per unit of time after a following medication of a specific dose of the drug, resulting from four kinetic processes: absorption, distribution, metabolism, and elimination. The pharmacokinetic and tissue distribution of Enrofloxacin were investigated in Nile tilapia (Oreochromis niloticus) following oral administration of a single dose of 12.5 mg/kg body weight (mixed with feed). Plasma and tissue samples, including liver and muscle, were collected at predetermined time points (0.25, 0.5, 1, 2, 4, 8, 12,16, 24 hours post-dosing). Enrofloxacin concentrations were determined using High Performance Liquid Chromatography (HPLC). The maximum concentrations (Cmax) in plasma, liver, and muscle were 1.1527, 19.7427, and 4.3230 μg/ml, respectively, with corresponding times to reach these concentrations (Tmax) of 4, 8, 8 hours, these results indicate widespread distribution of Enrofloxacin in tissues. After oral medication, Enrofloxacin concentrations in plasma and tissues were above the Minimum Inhibitory Concentration (MIC) for Aeromonas hydrophilla bacteria isolated from Nile tilapia (MIC = 0.060 mg/L). Based on Pharmacokinetic/Pharmacodynamic (PK/PD) index calculations for fluoroquinolone antibiotics using AUC/MIC or Cmax/MIC ratios, the Enrofloxacin regimen of 12.5 mg/kg body weight exhibited a positive therapeutic effect against Aeromonas hydrophilla infection.