Laboulbeniopsis termitarius (Thaxt) and Antennopsis gallica (Buchli and Heim) are two of the most common ectoparasitic fungi found on the body surface of termites. While visual observation under a dissecting microscope is a common method used to screen for such fungi, it generally requires a large number of termites and is thus very time consuming. In this study, we develop a fast, efficient protocol to detect fungal infection on the termite Reticulitermes speratus (Kolbe). Species-specific primers were designed based on sequence data and amplified using a number of universal fungus primer pairs that target partial sequences of the 18s rRNA gene of the two fungi. To detect these fungi in a robust yet economic manner, we then developed a multiplex nested polymerase chain reaction assay using species-specific primers. Results suggested that both fungi could be successfully detected, even in cases where L. termitarius was at low titer (e.g., a single thallus per termite).The new method described here is recommended for future surveys of these two fungi, as it is more sensitive, species specific, and faster than visual observation, and is likely to facilitate a better understanding of these fungi and their dynamics in host populations.