A rapid and accurate diagnosis of invasive fungal infections (IFIs) has been a great challenge particularly in cases requiring prompt antifungal treatment. In this study, four primer pairs were designed for a quadruplex PCR assay, which was developed for detection of four fungal species simultaneously. DNA extraction of cultured colonies and spiked blood samples were performed using conventional (phenol-chloroform) techniques and commercial DNA extraction kit. The optimum annealing temperature for this assay was 60°C. The assay was able to amplify all four genes and showed 100% specificity. No amplification of any genes was obtained against other species (n=14), which included two bacteria species. In conclusion, this quadruplex PCR assay is specific, rapid and reliable to detect A. fumigatus, A. terreus, C. albicans and C. glabrata simultaneously.