In patients with prolonged episodes of neutropenia, infections are associated with significant mortality. The exact diagnostic yield of blood cultures in this high-risk population is still unclear. To assess the yield of blood cultures, the spectrum of pathogenic organisms and the influence of blood culture results on the therapeutic management, we retrospectively evaluated the results from 2520 blood cultures obtained from 126 consecutive patients with high-risk neutropenia. Bacterial pathogens were detected in 219 blood culture samples (8.7%) of which 172 were Gram-positive and 47 were Gram-negative bacteria. Fungal pathogens were found in 13 blood cultures. A higher rate of Gram-positive pathogens and of fungi was found in patients with central venous catheters. Pathogens were detected in 14.3% of blood cultures obtained before the institution of antibiotic treatment and in 7% of blood cultures obtained under antibiotic treatment. Treatment was modified in 116/232 (50%) of positive blood culture findings. In patients with high-risk neutropenia, blood cultures are a valid diagnostic tool, both in antibiotic-naïve patients and in patients receiving antibiotic treatment, and provide important information for clinical decision making. The epidemiological data obtained are helpful for selecting empirical antibiotic treatment regimens.