A total of 287 faecal specimens of captive exotic birds from the orders Psittaciformes, Passeriformes and Columbiformes were randomly collected from Bohemian pet stores, avian breeders and avian keepers and were screened for the presence of human pathogenic microsporidia by polymerase chain reaction (PCR). Microsporidial DNA was identified in 115 faecal samples (40.1%). Single-species infection was detected in 36 birds (12.5%) for Enterocytozoon bieneusi, 36 birds (12.5%) for Encephalitozoon cuniculi and 18 birds (6.3%) for Encephalitozoon hellem. No Encephalitozoon intestinalis positive samples were identified. Moreover, co-infections were detected in 25 birds: E. bieneusi together with E. cuniculi in 14 animals (4.9%) or E. hellem in 11 cases (3.8%). E. hellem was present in 1A (5.2%) and three (0.3%) genotypes, E. cuniculi in I (2.4%), II (8.0%) and III (0.7%) genotypes and E. bieneusi in A (8.4%) and EbpA (10.8%) genotypes. Several of these genotypes have never been recorded in birds before. The results of this report suggest the low host specificity of E. bieneusi, E. hellem and E. cuniculi and describe 44 new avian hosts.