Bovine mastitis caused by bacterial pathogens, such as Staphylococcus (S.) aureus and Escherichia (E.) coli, is a major economic problem in dairy industry. In order to limit the presence of multi-resistant bacteria in bovine mastitis, alternatives for the treatment with antibiotics are urgently needed. Antimicrobial peptides (AMPs) have recently been discussed as a potential new strategy against bacterial infections. They are key players in the innate immune system, as they can directly act against microorganisms or modulate the immune system. The aim of our study was to test S. aureus and E. coli mastitis isolates for their susceptibility to the bovine cathelicidins, BMAP-27 and BMAP-28. Susceptibility testing was performed in analogy to the broth microdilution criteria described by the Clinical and Laboratory Standard Institute (CLSI) to determine MICs of 50 clinical S. aureus and 50 clinical E. coli isolates for BMAP-27 and BMAP-28. Based on the repetitive testing of four well-selected reference strains, the homogeneity of MIC variances for each peptide as well as the effect of temperature, oxygen level and plastic polymers on MIC testing was determined. Statistical analysis revealed not only strong peptide-specific variances, but also strain-specific variances in the technical procedure. Finally, using this technique, susceptibility testing of the field isolates revealed statistically significant peptide-specific differences in the MICs. While BMAP-27 showed lower MICs for E. coli, BMAP-28 showed lower MICs for S. aureus. However, these results clearly illustrate the need of susceptibility testing of AMPs on several unrelated strains and not only on one selected test organism.