The Eurasian beaver (Castor fiber) is an expanding species in Europe in recent decades due to reintroductions and natural population growth. Beavers expanded rapidly in the second half of the 20th century, and their expansion was particularly rapid in the Danube basin. Nowadays, the majority of the continuous population located in the central and eastern parts of the continent and a large disjunct population in Norway and Sweden. Despite the increasing population size, the role of the beaver as a source of waterborne pathogens is not firmly established or is often inferred from circumstantial data. In order to extend knowledge about the composition of the parasite fauna of beavers occurring in Slovakia, 21 faecal samples taken near their burrows from three sites (located in the Topľa, Poprad and Danube river basin) were examined microscopically and by polymerase chain reaction (PCR). PCR-positive specimens were further examined by DNA sequencing. Parasites were detected in 21% of the examined beavers, specifically the protozoa Cryptosporidium spp. (n = 2), Blastocystis sp. (n = 1), and microsporidia Enterocytozoon bieneusi (n = 1) and Encephalitozoon spp. (n = 1). Using the sequence analysis, two variants of Cryptosporidium proliferans, a new subtype of Blastocystis sp., genotype D of E. bieneusi and Encephalitozoon intestinalis were identified. A putatively novel Blastocystis subtype (ST), originated from a site near the Danube river (southwestern Slovakia), was proposed based on high genetic divergence from the closest described subtype ST12 (11.9%) and unique phylogenetic position in a clade composed of ST's 35–38. The increased risk of zoonotic transmission or transmission to other animals was particularly evident in the site near the Topľa river (northeastern Slovakia), where fungal spores of zoonotic genotype D of E. bieneusi and E. intestinalis, together with oocysts of the potentially zoonotic C. proliferans, were found.