In recent years, the absence of acquired antimicrobial resistance has become an important criterion to evaluate the biosafety of lactobacilli used as industrial starter or probiotic cultures. At present, however, standards for susceptibility testing of Lactobacillus strains or approved guidelines for interpreting the test results are not available. Hence, this study was carried out to contribute to the establishment of a standardized procedure for antimicrobial susceptibility testing of lactobacilli. The results obtained by testing 104 strains of the Lactobacillus acidophilus group were compared based on broth microdilution, disk diffusion, and Etest. Except for some specific agent-related effects, agreement between MICs resulting from the broth microdilution method and the Etest was good. In addition, inhibition zone diameters determined with disk diffusion correlated well with MICs from Etest and broth microdilution.