Background: Although nitrofurantoin has been used for > 60 years for the treatment of uncomplicated urinary tract infections, its pharmacodynamic properties are not fully explored. Use is increasing because of increasing resistance to other antimicrobials due to ESBLs. Methods: We tested nine ESBL+ and two ESBL- strains in time-kill assays. Bactericidal activity and regrowth were assessed for all species and concentrations. Early-phase pharmacodynamics was analysed with a sigmoidal E-max model and the maximal killing rate, slope and EC50/MIC ratio were determined for each species. Results: A bactericidal effect was found at a parts per thousand yen2x MIC for Enterobacter cloacae after 4-8 h, for Klebsiella pneumoniae after 8-10 h and for Escherichia coli after 12-16 h. Overall, no killing was observed at low sub-MIC concentrations, whereas regrowth was found at 0.5-1x MIC after a short decline in cfu. The lowest maximal killing rates were observed for E. coli (0.21aEuroS +/- aEuroS0.05 h(-1)), followed by K. pneumoniae (0.37aEuroS +/- aEuroS0.09 h(-1)) and E. cloacae (0.87aEuroS +/- aEuroS0.01 h(-1)). Surprisingly, the Hill slopes for these three species were significantly different (10.45aEuroS +/- aEuroS9.37, 2.68aEuroS +/- aEuroS0.64 and 1.01aEuroS +/- aEuroS0.06, respectively), indicating a strong concentration-dependent early-phase antibacterial activity against E. cloacae. EC50/MIC ratios were significantly lower for E. coli (0.24aEuroS +/- aEuroS0.08 mg/L) and K. pneumoniae (0.27aEuroS +/- aEuroS0.03 mg/L) as compared with E. cloacae (0.77aEuroS +/- aEuroS0.18 mg/L). Conclusions: Nitrofurantoin was bactericidal against all species, demonstrating an unusual differential pattern of activity with concentration-dependent-type killing behaviour against E. cloacae and time-dependent killing behaviour against E. coli, which may have significant consequences on species-dependent dosing regimens. The results also demonstrate that the pharmacodynamic properties of some drugs cannot be generalized within a family, here the Enterobacteriaceae.