This study investigated the in vitro bactericidal activity of an intramammary drug product by comparing the kill kinetics of cefalexin and kanamycin, alone and in fixed ratio combination, against Streptococcus uberis, Staphylococcus aureus and Escherichia coli strains isolated from field cases of bovine mastitis. The effect of milk as a diluent on the rate of bacterial killing was also assessed. Antibacterial kill kinetics was determined against each bacterial strain in Mueller-Hinton broth (MHB) and in milk. In MHB, the fixed cefalexin : kanamycin combination (1.5 : 1 w/w) exhibited a clear synergistic bactericidal activity against the strains tested. The combination also showed an enhanced killing activity in milk, as compared to either agent alone. The data show the occurrence of synergistic interactions between cefalexin and kanamycin, resulting in a faster and enhanced bactericidal activity against major mastitis pathogens. The study demonstrated that the combination exhibited a larger and faster rate of kill of S. aureus, S. uberis and E. coli compared to either cefalexin or kanamycin alone, while using a lower total amount of antibiotic. Synergistic and additive effects were also observed when milk was used as a medium. The results support the use of this combination of narrow spectrum antibiotics to treat clinical mastitis via the intramammary route and provide data on its killing kinetics.