The present study aimed to molecularly diagnose microsporidia in Nile tilapia and African catfish at the Qalyubia Governorate in Egypt and describe the histopathological alterations that are related with them. Seventy-nine out of 400 (19.75%) Nile tilapia, and forty-eight out of 200 (24%) African catfish had white nodules in their liver and kidneys were collected from natural water resources. BLAST analysis showed that 18S rDNA of isolated Glugea anomala from Nile tilapia (acc. no. OR766288) and from African catfish (acc. no. OR766287) revealed 100% sequence similarity with Glugea anomala (AF044391, AB923879) and Glugea arabica (KT005391). The highest seasonal infection rates with Glugea anomala in Nile tilapia and African catfish were recorded in autumn (32%) and spring (36%) respectively. Histopathological examination revealed the presence of microsporidian spores in the cytoplasm of degenerated and necrotic hepatocytes and renal tubular epithelium. On conclusion, the study recorded Glugea anomala in naturally infected Nile tilapia and African catfish that negatively alter the tissue and hence fish health status.