Seven hundred and eighty seven random milk samples were collected from cows and buffaloes at different localities and farms in Assiut Governorate. These samples represented by 379 and 408 quarter milk samples from 105 cows and 105 buffaloes, respectively. Animal-wise incidence of Subclinical Mastitis (SCM) based on California Mastitis Test (CMT) and Modified Whiteside Test (MWST) were 59.05 & 60.95% positive in cows and 33.33% & 33.33% positive in buffaloes, respectively. The animal-wise incidences of SCM in cows & buffaloes by bacteriological and mycological examinations were 60.95 & 28.57% and 34.29 & 0.95% positive, respectively. The most common bacteria causing SCM in cows were Staph. aureus, coagulase negative staphylococci (CNS), Str. pyogenes, Str. agalactiae, Str. dysgalactiae, E. coli, Klebsiella pneumoniae, Citrobacter diversus and Proteus mirabilis, respectively. While, in buffaloes were Staph. aureus, CNS, Str. pyogenes, Str. dysgalactiae and Corynebacterium bovis. Additionally, the most common yeasts and molds causing SCM in cows were Candida albicans, Candida tropicalis, Candida krusei, Candida sp., Geotrichum candidum, Aspergillus niger, Cladosporium cladosporioides, Fusarium proliferatum, Penicillium duclauxi, Rhodotorula sp., Stachybotrys elegans, near to Pyssochlamys nivea, Alternaria alternata, Stemphylium botryosum, Thermoascus aurantiacus, Trichosporon cuteanum and sterile mycelium. However, Phialophora sp. was only isolated from buffaloes in a percentage of 0.25%.