An effective multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of three major pathogens, Nosema bombycis Nägeli (Microsporidia: Nosematidae), Bombyx mori nucleopolyhedrovirus (Baculoviridae: genus Alphabaculovirus) (NPV), and Bombyx mori densovirus (Parvoviridae: genus Iteravirus) (DNV), in silkworms (Bombyx mori (Linnaeus); Lepidoptera: Bombycidae) was developed in this study. Polymerase chain reaction and real-time PCR tests and basic local alignment search tool searches revealed that the primers and probes used in this study had high specificities for their target species. The ability of each primer/probe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 8.5×103 copies for N. bombycis and Bombyx mori NPV (BmNPV) and 8.5×104 copies for Bombyx mori DNV (BmDNV). When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity, and high-throughput capabilities. Under the optimal volume ratio for the three primer/probe sets (3:2:2=N. bombycis:BmNPV:BmDNV), the multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation using DNA templates of the three pathogens in various combinations from individually infected silkworms; the early detection of N. bombycis was possible by day 3 post inoculation using the DNA isolated from the midgut of N. bombycis-infected silkworms.