Abstract: The aim of this study was to investigate in vitro the pharmacodynamics of Enrofloxacin (ENR) against an Escherichia coli gyrA mutant (E. coli MT/128). Broth microdilution testing was used to determine the Minimum Inhibitory Concentration (MIC) amd multi-step resistance selection was performed until reaching 1 μg mL•1 MIC. For time-kill experiments, colony counts were determined by plating each diluted sample onto Plate Count Agar and an integrated pharmacokinetic/pharmacodynamics area measure (log ratio area) was applied to all cfu data. A single C-T mutation was found in gyrA at codon 83. Concentration-dependent bacterial killing was observed for E. coli MT/128. Bactericidal activity for this strain was achieved within 4 h at concentrations • 8 times the MIC with no significant regrowth by 24 h.