Subclinical mastitis causes most economic loss in the dairy industry due to decreased milk quality and quantity. The goal of this study was to isolate and identify the mould and yeast from subclinical mastitic buffalo milk samples in Menoufia and Qalyubia Governorates. A total of 160 milk samples were collected and analyzed using the California mastitis test for the detection of subclinical mastitis. Seventy-five samples (46.88 %) tested positive for subclinical mastitis, indicating that subclinical mastitis is common in this area. Different mould and yeast from 55 subclinical mastitic milk samples (73.33%), could be isolated with ten samples (18.18%) demonstrating mixed fungal infection, indicating that fungal subclinical mastitis is spreading in these areas. Candida albicans (43.64 %), Penicillium spp, and Aspergillus fumigatus (38.18 % and 25.45 %, respectively) were shown to be the most common fungi linked with subclinical mastitis. Low percent identified yeast Rhodotorula and Aspergillus flavus 7.27and 1.81, respectively. The current results pointed to the necessity for field-based screenings of fungal subclinical mastitis and the development of safe methods for reducing fungal infections in order to provide consumers with high-quality milk and reducing the economic loss caused by fungal mastitis.