OBJECTIVES. To assess whether targeted short-term fluconazole prophylaxis reduces late-onset (>3 days of age) invasive fungal infection (IFI) among very low birth weight infants and extremely low birth weight (ELBW) infants and to assess mortality rates, toxicity, and costs associated with this intervention. METHODS. An observational study of 2 subsequent epochs of inborn infants with birth weight of <1500 g or gestational age of 3 days. RESULTS. IFI was observed for 13 (6.3%) of 206 infants in the control epoch and 2 (1.1%) of 178 in the fluconazole epoch, with a common odds ratio of 0.166. Logistic regression analysis taking into account all published factors (except for fungal colonization) showed that the fluconazole epoch was associated significantly with lower IFI rates. We observed no change in late (>3 days) mortality rates (11 of 206 infants in the control epoch vs 8 of 178 infants in the prophylaxis epoch). The mortality rate for ELBW infants with IFI was low (15%) in our study. Fluconazole was administered to 81% of ELBW infants, who received a median of 8 doses, and 41% of larger infants, who received a median of 5 doses. The intervention was cost-effective, and the effective number needed to treat to prevent 1 IFI was 10. CONCLUSIONS. This study suggests that targeted short-course fluconazole prophylaxis in very low birth weight and ELBW infants may be efficacious and cost effective.