Bitter Rot (Greeneria uvicola) and Ripe Rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in sub-tropical grape growing regions of Australia where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute towards their effective management and control, a rapid and reliable species-specific real-time PCR method was developed based on the polymorphic portion of the internal transcribed spacer (ITS) region of the two fungi. It was found that within 6 to 8 hours post inoculation, the assay could detect as little as 20 fg genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.