The pharmacokinetics aspects of marbofloxacin were studied in healthy lactating buffaloes after single intravenous (IV) and single intramuscular (IM) injections at a dose of 2 mg/kgb.wt. Drug concentration in blood, milk and urine were determined by microbiological assay. After IV injection, marbofloxacin serum concentration– time curves were characteristic of a two-compartment open model. The distribution and elimination half-lives (T0.5α, T0.5β) were 0.31 and 4.74 h respectively. Following IM injection, peak serum concentration (Cmax) of 1.49 μg ml-1 was attained at 1.36 h. The absorption and elimination half-lives (T0.5ab, T0.5el) were 0.43 and 3.27 h respectively. The systemic bioavailability of the IM administration (F %) was 99.39%. Following IV and IM administration, the drug penetration into the milk was rapid and extensive with marbofloxacin concentration exceeding those of serum. It was eliminated from the milk by slower rate than that of the serum and the drug was not detected in milk 24h after treatment. Marbofloxacin was detected in the urine in a high concentration than that of serum and reach high level in urine 1 h after administration then decline until 24 h. So marbofloxacinis suitable and effective for injection to buffaloes by a dose of 2 mg/kg every 12 h for treatment of mastitis and urinary tract infection caused by sensitive organism.