Nine small-scale dairy farms include 982 animals (Friesian cows, 565 and buffaloes, 417) at Kaluobia and Menofia Governorates were clinically inspected for detection of udder and/or milk abnormalities, and the apparently healthy animals were subclinically tested by California mastitis test to indicate the prevalence of clinical and subclinical mastitis during one lactation season; Nov., 2007 – Dec., 2008). The isolated bacterial pathogens were identified by conventional culturing procedures whereas the isolated Mycoplasma was molecularly identified using Polymerase Chain Reaction (PCR). The current results indicated that overall prevalence of clinical and subclinical mastitis in cows' farms was 9.56 % and 27.61 %, respectively, and in buffaloes' farms was 6.00 % and 17.50 %, respectively. The high susceptible rate (P < 0.01) of cows to mastitis than buffaloes was discussed. Staphylococcus aureus was the predominant mastitis pathogen of clinical and subclinical mastitis in dairy cows and buffaloes. Mycoplasma could not be isolated from buffaloes. In Friesian cows, Mycoplasma occupied a considerable level of the isolated contagious mastitis pathogen from the clinically mastitic cases (19.70 %) and it occupied the second grade (28.57 %) following Staphylococcus aureus (42.86 %) as etiologic agent responsible for incurable mastitis of cows. Twenty-eight isolates of Mycoplasma were biochemically determined and 17 of them were identified as Mycoplasma bovis by PCR, which is easier and more flexible tool than conventional culturing procedures. In-vitro antibiotic-disc-susceptibility to Staphylococcus aureus was achieved. The culturally and/or molecularly (PCR) positive cases of Mycoplasma mastitis, in particular Mycoplasma bovis mastitis, should culled as early out the dairy herd to avoid the lateral transmissions of infection to neighboring unaffected animals, and to avoid the transmission of infection to neonates and calves. Mycoplasmal examination, in the infected farm, should regularly do in paralleling with routine detection of mastitis until elimination by segregate/cull the infected cases.