Ancient DNA (aDNA) of Encephalitozoon intestinalis (Microsporidia, Fungi) was detected in archaeological material originated from New Town of Prague (Czech Republic) with the use of molecular methods. Microsporidial aDNA was found in 3 samples originating from 2 objects, in a well/cesspit (samples from layers from the 18th century) and in a well from the 18th/19th century. The ability to use molecular methods to detect microsporidia extends the range of paleoparasitological inquiry, and could contribute to a better understanding of parasites shared between human and animals.