Puerperal metritis in cows is often treated with antibacterial drugs. The prudent use of antibiotics in farm animals requires state-of-the-art knowledge of their pharmacokinetics and data from sensitivity tests of pathogenic bacteria. Changes in oxytetracycline levels in the uterine secretion over time after intrauterine administration in cows with metritis were evaluated in relation to the sensitivity of pathogenic bacterial isolates. Oxytetracycline levels in plasma, milk and uterine secretion were determined via LC-MS/MS analysis. Pathogenic bacteria were isolated and their sensitivity to oxytetracycline was determined. The concentrations of oxytetracycline in the uterine secretion were 433.79 (39.17-1668.76) µg·mL-1 six hours after the third application at a dose of 8 mg/kg and 84.33 (1.58-467.55) µg·mL-1 96 h after the last treatment. These levels were higher than the minimum inhibitory concentrations-namely, between 4 and 64 µg·mL-1-against pathogenic isolates Trueperella pyogenes, Streptococcus intermedius, Escherichia coli and Bacillus pumilis. Higher concentrations over time were measured in milk rather than in plasma, indicating the need to monitor the withdrawal time for milk. The intrauterine infusion of oxytetracycline for three consecutive days resulted in the successful treatment of metritis in terms alleviating inflammation and restoring the estrus cycle in cows.