The efficacy and field safety of marbofloxacin administered as a single-dose treatment to lactating dairy cows, suffering from acute mastitis (associating general and local clinical signs) were evaluated in a masked, randomised, European, multicentre study. A total of 354 lactating dairy cows suffering from acute untreated mastitis (in only one quarter) were enrolled and treated on day 0 with either marbofloxacin (10 mg/kg, intramuscular administration; n = 178) or danofloxacin (6 mg/kg, subcutaneous administration; n = 176). Milk was collected for microbiological evaluation prior to any treatment. Results showed that 194 cows were positive for E. coli, of which 148 were fully compliant with the study protocol (marbofloxacin n = 72 and danofloxacin n = 76). E. coli positive cattle were clinically assessed on days 1, 2, 3, 7, 15 and 27 following treatment and milk was sampled on days 15 and 27. Success rates (cure + clear improvement) were compared using a non-inferiority margin analysis approach. Both treatments were well tolerated, a high success rate was observed on day 15, and marbofloxacin was non-inferior (P < 0.00001) to danofloxacin (day 15 success rate, marbofloxacin vs. danofloxacin: 86.1% vs. 81.6%). Secondary parameters (time course of the clinical outcome, bacteriological cure rate on days 15 + 27, global cure rate (clinical + bacteriological), relapse rate) confirmed that there was no difference between treatments (P >= 0.33), indicating a consistent although non-significant trend in favour of a better efficacy of marbofloxacin. In conclusion, a single injection of marbofloxacin is safe and efficacious for the treatment of acute E. coli mastitis.