Ten isolates of the recently delineated genospecies Borrelia spielmanii were tested against antimicrobial agents used to treat Lyme disease and compared to eight isolates of the other three human-pathogenic borrelial genospecies. Despite some small but significant differences in four out of eight antibiotic agents, the susceptibility pattern of B. spielmanii mainly parallels that of the other known human-pathogenic members of the B. burgdorferi sensu lato complex.