Abstract Cryptosporidium spp. and microsporidia are opportunistic parasites affecting a wide range of hosts in which they can be potentially life threatening in immunocompromised individuals. Diagnosis usually relies on the identification of the stained Cryptosporidium oocyst or microsporidial spores, but these methods lack sensitivity and require highly trained technicians to perform and interpret the results. Molecular diagnosis offers an alternative with both superior sensitivity and specificity as compared to microscopy. Although replacing microscopy with nucleic acid based methods is hampered by the higher costs, in particular in developing countries, multiplexing the detection of more than one parasite in a single test has been found to be very effective and would decrease the cost of the test without the need for new equipment, as it would be the case for quantitative PCR. The method shown in this report for the simultaneous detection of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis by multiplex nested PCR, has proved to have several advantages versus microscopy such as higher sensitivity and specificity, low subjectivity and a minimal need for specialist's training to interpret the results. The present multiplex assay can fill an important gap to identify other possible causative agents of several diarrheal diseases which until present remain undiagnosed and can improve the epidemiology of the disease with a more reliable detection method.