High mortality of nestling cockatiels (Nymphicus hollandicus) was observed in one breeding flock in Slovakia. The nestling mortality affected 50% of all breeding pairs. In general, all the nestlings in affected nests died. Death occurred suddenly in 4- to 6-day-old birds, most of which had full crops. No feather disorders were diagnosed in this flock. Two dead nestlings were tested by nested PCR for the presence of avian polyomavirus (APV) and Chlamydophila psittaci and by single-round PCR for the presence of beak and feather disease virus (BFDV). After the breeding season ended, a breeding pair of cockatiels together with their young one and a fledgling budgerigar (Melopsittacus undulatus) were examined. No clinical alterations were observed in these birds. Haemorrhages in the proventriculus and irregular foci of yellow liver discoloration were found during necropsy in the young cockatiel and the fledgling budgerigar. Microscopy revealed liver necroses and acute haemolysis in the young cockatiel and confluent liver necroses and heart and kidney haemorrhages in the budgerigar. Two dead cockatiel nestlings, the young cockatiel and the fledgling budgerigar were tested positive for APV, while the cockatiel adults were negative. The presence of BFDV or Chlamydophila psittaci DNA was detected in none of the birds. The specificity of PCR was confirmed by the sequencing of PCR products amplified from the samples from the young cockatiel and the fledgling budgerigar. The sequences showed 99.6-100% homology with the previously reported sequences. To our knowledge, this is the first report of APV infection which caused a fatal disease in parent-raised cockatiel nestlings and merely subclinical infection in budgerigar nestlings.