The present study was carried out on 120 examined cultured Nile tilapia, Oreochromis niloticus (O. niloticus) (80 diseased fish and 40 apparently healthy) were collected alive from private fish farms at Kafr El Sheikh Governorates from April to October 2018. Grossly, examined diseased O. niloticus revealed eroded fins, redness on the skin, congested protruded anal opening and sometimes darkness on skin, ulcers with different sizes, and eye turbidity especially in fish naturally infected with Aspergillus, Fusarium and Pencillium. Mycological examination revealed isolation of 7 Genera of fungi from total incidence of 300 fungal isolates; 240 fungal isolates from diseased O. niloticus and 60 isolates from apparently healthy one. Aspergillus flavus and Aspergillus niger were the most predominant fungi isolated from either apparently healthy or even diseased one mainly from internal organs. Molecular identification was performed using inter-transcriped spacer (ITS) gene for A. flavus and A. niger. Developed PCR assay specific primer detect A. flavus and A. niger showing clear bands at 595 and 600 bp molecular weight, respectively.