Antifungal prophylaxis is increasingly used in very low birth weight (VLBW) infants who are at risk for severe fungal infections. Our objective was to assess the effectiveness of targeted fluconazole prophylaxis for high-risk VLBW infants. A retrospective cohort study with historical controls was performed. During the period 2007-2008, all high-risk VLBW infants (birth weight, ≤1,000 g; gestational age, ≤28 weeks; seven antimicrobial therapy or additional risk factors present) received fluconazole prophylaxis until risk factors were not present. Treated infants were compared to a gestational age- and birth weight-matched untreated cohort. Statistical analyses used univariate and multivariate analyses. The main outcome variable was a breakthrough fungal bloodstream infection (BSI). The prophylaxis cohort of 130 VLBW infants was compared to 319 control infants. The rate of fungal infections was significantly lower in the fluconazole prophylaxis group (1 of 130 vs. 19 of 319, p = 0.016); however, they did not differ in mortality (16.2 vs. 15 %, p = 0.77) or complications of prematurity. Fluconazole prophylaxis was associated with a significant decrease in candidal BSI (odds ratio, 0.05; 95 % confidence interval, 0.005-0.523). Selective vs. nonselective prophylaxis reduced the number of infants treated from 247 to 130. Conclusion Targeted fluconazole prophylaxis in VLBW infants is effective in preventing fungal infections without increasing the risk of BSI among low-risk infants.