A prospective observational study of invasive candidiasis was conducted in the neonatal intensive care unit of Aristotle University in Hippokration Hospital between 1994 and 2000. During this period, 59 neonates developed invasive candidiasis (58 cases of candidemia and 1 case of peritonitis), resulting in an overall incidence of 1.28% that showed a decreasing trend over the study period. Eleven (18.6%) cases developed within the first week of life and the others within a mean (+/-SEM) of 13.4+/-1.7 days after birth. The three most frequent causative species were Candida albicans (65.5%), Candida parapsilosis (15.5%), and Candida tropicalis (7%). C. albicans was the predominant species between 1994 and 1998, whereas, non-albicans Candida spp., particularly C. parapsilosis, were the most frequent species during the period 1999-2000 (P<0.001). While the overall mortality due to candidemia was 29% (17 of 59 cases), mortality associated with C. albicans and C. parapsilosis was 39.5% and 11.1%, respectively (P=0.032), and that observed in the 1999-2000 period was 0% (P=0.011). Virtually all isolates were susceptible to amphotericin B, flucytosine, fluconazole, and itraconazole, and no increases in minimal inhibitory concentrations were observed during these years. With the exception of a limited cluster of cases due to genotypically identical isolates, no clonal relation of C. albicans isolates was found. Moreover, no clonal persistence of C. albicans and no decrease in antifungal drug susceptibility occurred over the 6-year study period. Non-albicans Candida spp., mostly C. parapsilosis, have emerged as important pathogens in neonatal intensive care units, with infected patients having better outcomes as compared to patients infected with C. albicans.