The minimal inhibitory concentrations (MICs) of 23 Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis (PDD) lesions in Japan were investigated by a broth microdilution method using 15 antimicrobial agents. Although all MIC values showed a monomodal distribution, the MICs of the antimicrobial agents for 90% (MIC₉₀) of the isolates tested varied among the agents examined. The MIC₉₀ values for penicillin G, ampicillin, and erythromycin were <0.06 μg/ml. In contrast, the MIC₉₀ values for kanamycin, streptomycin, rifampicin, sulfamethoxazole, trimethoprim, and colistin were >128 μg/ml. Oxytetracycline, lincomycin, enrofloxacin, chloramphenicol, ceftiofur, and gentamicin showed intermediate values, i.e., 0.5~32 μg/ml. The present study suggested that no isolate had acquired resistance to the antimicrobial agents examined, although they may have natural resistance to some agents. Furthermore, the in vitro antimicrobial susceptibility data would provide helpful information for PDD treatment and the development of a selective medium for isolating the organism effectively.