Real-time PCR provides a fast, reliable, and cost-effective method for detecting the presence or absence of Petri disease fungi in grapevines. The primer pairs, Pmo1f + Pmo2r, and Pac1f + Pac2r, were designed for species and genus-specific amplification of Phaeomoniella chlamydospora and Phaeoacremonium spp. respectively, using realtime PCR with SYBR® Green. The primers were specific and showed no primer-primer dimers until after 35 cycles. Pa. chlamydospora was detected in roots, shoots, and young trunks of drill-inoculated vines. Phaeoacremonium was detected in trunk cross-sections of naturally infected vines from which Phaeoacremonium aleophilum had been isolated. The protocol presented here can be adapted to provide a reliable detection system for research and industry.