Pharmacodynamic and mutant prevention properties of the fluoroquinolone pradofloxacin (PRA) were measured against a set of 17 Escherichia coli strains carrying no, one or two known mutations conferring reduced fluoroquinolone susceptibility. The strains included susceptible wild-types, isogenic constructed mutants, isogenic selected mutants and clinical isolates. The effectiveness of PRA was determined with regard to preventing the selection of resistant mutants, using static and changing concentrations of drug. Ciprofloxacin was used as a reference drug. Minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of PRA for the susceptible wild-type strains were in the range 0.012–0.016 mg/L and 0.2–0.3 mg/L, respectively, giving a mean ± standard deviation mutant prevention index (MPI = MPC/MIC) of 17.7 ± 1.1. The mean MPI PRA of the 14 mutant strains was 19.2 ± 12, and the mean MPI across all 17 strains was 18.9 ± 10.8. In an in vitro kinetic model in which PRA was diluted with a half-life of 7 h to mimic in vivo conditions, an initial concentration of PRA of 1.6–2.4 mg/L (8–10× MPC), giving a PRA AUC/MPC ratio of 73–92, and a T>MPC of 21–23 h was sufficient to prevent the selection of resistant mutants from the three susceptible wild-type strains. Dosing to reduce selection for antibiotic resistance in veterinary therapy has a role in reducing the reservoir of resistant mutants. We conclude that a level of dosing that prevents the selection of resistant mutants during therapy should be achievable in vivo