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Bats may carry various viruses and bacteria which can be harmful to humans, but little is known about their role as a parasitic source with zoonotic potential. The aim of this study was to test wild bats for the presence of selected parasites: <i>Toxoplasma gondii</i>, <i>Neospora caninum</i> and microsporidia <i>Encephalitozoon</i> spp. In total, brain and small intestine tissues of 100 bats (52 <i>Myotis myotis</i>, 43 <i>Nyctalus noctula</i> and 5 <i>Vespertilio murinus</i>) were used for the DNA isolation and PCR detection of the abovementioned agents. <i>Toxoplasma gondii</i> DNA was detected by real-time PCR in 1% of bats (in one male of <i>M. myotis</i>), while all bats were negative for <i>N. caninum</i> DNA. <i>Encephalitozoon</i> spp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-two <i>M. myotis</i>, two <i>N. noctula</i> and one <i>V. murinus</i>). Positive samples were sequenced and showed homology with the genotypes <i>Encephalitozoon cuniculi</i> II and <i>Encephalitozoon hellem</i> 2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity of <i>Encephalitozoon</i> spp. detected in bats.