The aim of this study was to compare the pharmacokinetics of marbofloxacin after intravenous (IV) administration of a single dose of 10 mg/kg to calves of different ages. The study was carried on 1- (n = 6), 2- (n = 6), and 4-month-old (n = 6) Montofon calves. Plasma concentrations of marbofloxacin were measured using HPLC, and pharmacokinetic data were calculated by non-compartmental analysis. The elimination half-life (t1/2ʎz), volume of distribution at steady state (Vdss), total clearance (ClT), and area under the concentration-versus time curve (AUC0–∞) values of marbofloxacin in 1-month-old calves were 10.62 h, 1.03 L/kg, 0.08 L/h/kg, and 127.90 h*μg/mL, respectively. While the t1/2ʎz (from 10.62 to 3.36 h) and AUC0–∞ (from 127.90 to 47.35 h*μg/mL) decreased in parallel with the age of the calves, ClT (from 0.08 to 0.21 L/h/kg) increased. The Vdss of marbofloxacin was higher in 1- and 2-month-old calves compared to 4-month-old calves. After IV administration of marbofloxacin at a dose of 10 mg/kg, an ƒAUC0–24/MIC90 ratio of ≥ 125 was obtained for bacteria with MIC90 values of ≤ 0.60, ≤ 0.39 and ≤ 0.27 μg/mL in 1-, 2-, and 4-month-old calves, respectively. These results show that the antibacterial effect of marbofloxacin, which has concentration-dependent activity, decreases due to age-related pharmacokinetic changes and that the 10 mg/kg dose should be reviewed according to the MIC90 value of the bacteria.