Invasive fungal diseases (IFDs) are a global threat and kill millions of people each year. Candida species are the most common cause of fungal blood stream infections and are associated with high rates of morbidity and mortality. Prompt administration of antifungals is critical for patient survival, however conventional techniques for fungal detection lack sensitivity and can take days for results. In our pilot study we investigated the applicability of using flow cytometry to rapidly detect Candida cells in simulated blood culture samples using different yeast cell markers. For assay development, 28 yeast isolates from 6 different Candida species were spiked into blood culture bottles (with donor blood) at a standardised concentration (1 × 105 yeast cells/mL) and then loaded into a BD Bactec™ FX automated blood culture incubator. Once samples were flagged as positive, human cells were lysed with 0.5% Triton-X and water and stained with human cell markers (CD235a/CD45) and combinations of yeast cell markers (Calcofluor White, Concanavalin A and a Candida albicans antibody) for 30 minutes prior to processing on the Attune NxT flow cytometer. We observed non-specific binding of Calcofluor White stain to unlysed white blood cells, however fluorescence enhancement (FE) levels were much higher when bound to yeast cells (average of 300 compared to 6.7). Average FE values of 264 and 250 were observed for Concanavalin A and the Candida albicans antibody respectively when bound to yeast cells. Using our assay, we were able to detect and enumerate all yeast isolates regardless of species in both our simulated samples and a clinical episode of Candida albicans candidemia. These findings highlight the potential of using flow cytometry as a rapid, high-throughput method for fungal detection in blood cultures.