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Objectives: We aimed to assess the performance of the Novodiag^® Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections. Methods: A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia. Results: Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for <i>Blastocystis hominis</i>, <i>Entamoeba histolytica</i> and <i>Cyclospora cayetanensis</i> but was lower for <i>Giardia intestinalis</i> (85.2%) and ≤50% for <i>Cystoisospora belli</i> and <i>Dientamoeba fragilis</i>. In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for <i>G. intestinalis</i>, <i>D. fragilis</i>, <i>Cryptosporidium</i> spp. and <i>E. histolytica.</i> Sensitivity was 100% for <i>Enterocytozoon bieneusi</i>, but none of the five samples containing <i>Encephalitozoon</i> spp. were detected. Conclusions: The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR.