We report an increased occurrence of fluconazole-resistant Candida parapsilosis after a 4-year period of antifungal prophylaxis in a premature animal neonatal intensive care unit. Although prevention of nosocomial fungal infections in premature infants is desirable, implementation of fluconazole prophylaxis should be undertaken with caution. Where such programs are in place, evaluation of fungal isolates for drug resistance should be considered.