Simple SummaryIt is well known that mammals can harbor various pathogens that can affect humans (known as zoonotic pathogens) including viruses, bacteria, fungi and parasites. Microsporidia are a group of pathogens related to fungi and parasites of several animals that can cause diarrhea or systemic infection in humans. Due to the limited knowledge about microsporidia infection in hedgehogs worldwide, this study aimed to analyze the presence and identity of microsporidia in a group of North African hedgehogs from the Canary Islands (Spain).Enterocytozoon bieneusiandEncephalitozoon cuniculi, two zoonotic species of microsporidia, were identified. These results suggest that microsporidia species with zoonotic risk circulate in the archipelago.AbstractMicrosporidia are unicellular eukaryotic obligate intracellular parasites with a wide range of hosts reported worldwide; however, little is known about the epidemiological data on microsporidia infection in animals from the Canary Islands. Since data on microsporidia infection in hedgehog species are scarce, the aim of this study was to analyze the presence and identity of microsporidia in a group of North African hedgehogs (Atelerix algirus) using microscopic and molecular methods. From December 2020 to September 2021, a total of 36 fecal samples were collected from naturally deceased hedgehogs from Tenerife and Gran Canaria. All samples showed spore-compatible structures (100%; 36/36) under microscopic analysis, of which 61.1% (22/36) were amplified via the nested-polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. After Sanger sequencing and ITS analysis,Enterocytozoon bieneusiwas detected in 47.2% (17/36) of the samples, identifying two novel genotypes (AAE1 and AAE2), followed by the detection of an undetermined species in 8.3% (3/36) andEncephalitozoon cuniculigenotype I in 5.6% (2/36) of the samples. This study constitutes the first report of microsporidia species inAtelerix algirusworldwide, highlighting the high prevalence of zoonotic species.