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:Toxoplasma gondii,Neospora caninumand microsporidiaEncephalitozoonspp. In total, brain and small intestine tissues of 100 bats (52Myotis myotis, 43Nyctalus noctulaand 5Vespertilio murinus) were used for the DNA isolation and PCR detection of the abovementioned agents.Toxoplasma gondiiDNA was detected by real-time PCR in 1% of bats (in one male ofM. myotis), while all bats were negative forN. caninumDNA.Encephalitozoonspp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-twoM. myotis, twoN. noctulaand oneV. murinus). Positive samples were sequenced and showed homology with the genotypesEncephalitozoon cuniculiII andEncephalitozoon hellem2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity ofEncephalitozoonspp. detected in bats.