Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) were evaluated of the antimicrobial chemotherapeutics amoxicillin, azithromycin, cefotaxime, ceftriaxone, doxycycline, penicillin G sodium, roxithromycin, and trimethoprim-sulfamethoxazole for 30 Borrelia strains from various sources (skin, cerebrospinal fluid, ticks). Of these strains 29 were Lyme disease agents of the species Borrelia afzelii (n = 12), Borrelia burgdorferi sensu stricto (n = 4), Borrelia garinii (n = 13), and one was the relapsing fever strain Borrelia turicatae (n = 1). Tests were performed in microtiter plates by broth dilution. MIC was determined after 72 hours of incubation by comparing growth control with the antibiotic dilutions by means of dark field microscopy. Strains tested were susceptible against amoxicillin, azithromycin, cefotaxime, ceftriaxone, doxycycline, and penicillin G sodium, partly susceptible to roxithromycin, and resistant to trimethoprim-sulfamethoxazole. No statistically significant differences in MIC and MBC were seen among the different antibiotics with the various Borrelia species.