Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. A total number of 150 random samples (100 of buffaloe`s milk and 50 of cow's milk) were collected from dairy farms and dairy shops in Assiut city as well as from some villages around the city. Physical examination of all samples revealed absence of any visible abnormalities. California Mastitis Test (CMT) was carried out on all samples to detect the subclinical cases. 59 % of buffaloes and 68 % of cow's milk samples were positive. The average count of total yeasts and mold counts was 3.29x10<sup>2</sup> and 3.59x10<sup>2</sup> in examined samples of buffaloes and cow milk, respectively. Samples were examined for the presence of Prototheca spp < /em>. 12 % of buffaloe`s milk samples were positive. On the basis of colony morphology and microscopic appearance, after incubation at 38^ºC for 48–72 hrs, Prototheca spp < /em>. were isolated, identified and sub-cultured for further identification. P. zopfi and P. wickerhamii were foud in 7 % and 5 % of the positive samples-respectively. In addition, Prototheca spp < /em>. was not isolated from any of examined cow's milk samples. Antibiotics sensitivity test was carried out to detect antimicrobial susceptibility of isolated microorganisms.