In order to have a diagnostic test for invasive candidiasis, an immunofluorescent assay (IFA) in buffy coat was developed and evaluated. This test was compared to buffy coat culture and blood culture. The study was divided into two parts: evaluation of the diagnostic test in a rabbit model, and for diagnosis in patients at high risk for invasive candidiasis. Part I: Rabbits were experimentally infected. Of 13 rabbits with disseminated candidiasis blood was drawn from central venous catheter and from peripheral veins. From catheters, Candida was detected in 13/13, 10/13 and 8/13, by buffy coat culture, IFA, and blood culture, respectively. From peripheral veins, Candida was detected in 10/10 by buffy coat cultures, in 6/10 by IFA and in 3/10 by blood cultures. The gold standard was histopathological diagnosis; sensitivity for IFA was 60% with peripheral blood and 76% with catheter blood samples. Part II: IFA and blood cultures were compared in clinical samples from hospitalized children. Forty patients were included. Candida was isolated from blood cultures in four patients whereas IFA detected Candida in 22 patients. Differences were statistically significant (p < 0.05). We conclude that IFA in buffy coat smears is a reliable test to detect candidemia, and would be a useful tool to help physicians with treatment decisions with amphotericin B.