Aim: To assess the bacterial killing rate produced by a combination of cefalexin and kanamycin at two different concentration ratios. Methods and Results: Time-kill kinetics of cefalexin and kanamycin, individually and in combination, were determined against one strain each of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. The combination was tested using two fixed ratios (cefalexin : kanamycin ratios of 1 center dot 25 : 1 and 1 : 2 center dot 3) and two concentrations of each ratio. Time-kill curves produced with either ratio were quite similar. Against most bacterial species, higher concentrations produced faster kill. In all cases, the combination of cefalexin and kanamycin showed faster and greater kill at lower antibiotic concentrations than those observed with either drug alone. Conclusions: The combination of cefalexin and kanamycin results in a fast initial killing of major mastitis pathogens at both concentration ratios. Significance and Impact of the Study: The combination of cefalexin and kanamycin achieved rapid bacterial kill at concentrations and ratios that can be achieved in vivo following intramammary infusion of a mastitis treatment.