Two microsporidian species infect honeybees worldwide, Nosema apis and Nosema ceranae. Two different clinical patterns are considered: nosemosis type A (N. apis) and nosemosis type C (N. ceranae). Nosemosis type A is characterized in acute forms and nosemosis type C shows no clear outward clinical signs. The development of a rapid and simple tool for Nosema detection could allow beekeepers or veterinarians to carry out diagnostic tests in situ. Currently, PCR and microscopy are expensive techniques that require qualified staff and may not be available in every laboratory. The present study describes the production and characterization of four monoclonal antibodies (mAbs) against N. ceranae and N. apis, and the development of an IFAT. An IFAT using the mAbs was compared with microscopy and PCR for 180 beehive samples. The diagnostic test revealed similar sensitivity and specificity percentages to IFAT (97.79% and 93.18%, respectively) and microscopy (97.79% and 95.45%), considering 100% for the PCR as the 'gold standard'. A mAb (7D2) was patented for its high specificity for N. ceranae. The IFAT using the mAbs is a good alternative to microscopy and PCR in laboratories where PCR is not available for the detection and identification of both Nosema spp.