Microsporidia are a cause of emerging and opportunistic infections in humans and animals. Although two drugs are currently being used to treat microsporidiosis, concerns exist that albendazole is only selective for inhibiting some species of microsporidia that infect mammals, and fumagillin appears to have been found to be toxic. During a limited sequence survey of the Vittaforma corneae (syn. Nosema corneum Shadduck, Meccoli, Davis et Font, 1990) genome, a partial gene encoding for the ParC topoisomerase IV subunit was identified. The purpose of this set of studies was to determine if fluoroquinolones, which target topoisomerase IV, exert activity against Encephalitozoon intestinalis (syn. Septata intestinalis Cali, Kotler et Orenstein, 1993) and V. corneae in vitro, and whether these compounds could prolong survival of V. corneae-infected athymic mice. Fifteen fluoroquinolones were tested. Of these, norfloxacin and ofloxacin inhibited E. intestinalis replication by more than 70% compared with non-treated control cultures, while gatifloxacin, lomefloxacin, moxifloxacin, and nalidixic acid (sodium salt) inhibited both E. intestinalis and V. corneae by at least 60% at concentrations not toxic to the host cells. These drugs were tested in vivo also, where gatifloxacin, lomefloxacin, norfloxacin, and ofloxacin prolonged survival of V. corneae-infected athymic mice (P < 0.05), whereas moxifloxacin and nalidixic acid failed to prolong survival. Therefore, these results support continued studies for evaluating the efficacy of the fluoroquinolones for treating microsporidiosis and for characterizing the target(s) of these fluoroquinolones in the microsporidia.