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Abstract Background Several clinical trials have demonstrated the efficacy of fluconazole as empiric antifungal therapy in cancer patients with fever and neutropenia. Our objective was to assess the frequency and resource utilization associated with treatment failure in cancer patients given empiric fluconazole antifungal therapy in routine inpatient care. Methods We performed a retrospective cohort study of cancer patients treated with oral or intravenous fluconazole between 7/97 and 6/01 in a tertiary care hospital. The final study cohort included cancer patients with neutropenia (an absolute neutrophil count below 500 cells/mm3) and fever (a temperature above 38°C or 100.4°F), who were receiving at least 96 hours of parenteral antibacterial therapy prior to initiating fluconazole. Patients' responses to empiric therapy were assessed by reviewing patient charts. Results Among 103 cancer admissions with fever and neutropenia, treatment failure after initiating empiric fluconazole antifungal therapy occurred in 41% (95% confidence interval (CI) 31% – 50%) of admissions. Patients with a diagnosis of hematological malignancy had increased risk of treatment failure (OR = 4.6, 95% CI 1.5 – 14.8). When treatment failure occurred the mean adjusted increases in length of stay and total costs were 7.4 days (95% CI 3.3 – 11.5) and $18,925 (95% CI 3,289 – 34,563), respectively. Conclusion Treatment failure occurred in more than one-third of neutropenic cancer patients on fluconazole as empiric antifungal treatment for fever in routine clinical treatment. The increase in costs when treatment failure occurs is substantial.