Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to : i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp; ii) give an update about the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performances of the assay were assessed, and genotyping of all cryptosporidia and microsporidia isolates was performed. The multiplex PCR sensitivity reached 1 copy/μl for each targeted pathogen. Copro-antigen testing for cryptosporidiosis diagnosis had a sensitivity of 73%. Microscopy sensitivity was 64% for cryptosporidia and 50% for microsporidia, compared to PCR. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species), 5 for E. bieneusi and 2 for E. intestinalis. The overall prevalence was 3.1% for cryptosporidia and 1.5% for microsporidia but reached 7.3% and 2.4% (6 and 2 patients), respectively, in kidney transplant patients (82 patients). Two cases of E. intestinalis infections were diagnosed in children returning from tropics. This study is the first to assess a multiplex qPCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalence of both pathogens were observed in kidney transplant recipients.Copyright © 2020. Published by Elsevier Inc.