A total of 80 milk samples randomly selected from milk samples collected from cows and buffaloes of a dairy mastitic herd at Giza Governorate in Egypt, manifesting clinical mastitis. Milk samples were submitted to examination for the presence of Mycoplasma infection by the culture method and ELISA test. Using the culture method, 40 cows milk samples harbored 10 Mycoplasma species, 7 of them were antigenically related to M. bovis and 3 were M. arginini, while 40 buffalo milk samples revealed 12 Mycoplasma isolates, 4 of them were M. bovirhinis 2 were M. bovis and 6 M. bovigenitalium. ELISA test was applied for the detection of Mycoplasma antigen in the milk samples, it was found that 12 cows milk samples were positive for Mycoplasma, 8 were M. bovis, 4 were M. arginini, while 14 buffalo milk samples were positive for Mycoplasma, 7 of them were M. bovig, 3 were M. bovis and 4 were M. bovirhinis. It was concluded that the number of Mycoplasma positive milk samples was 5 % higher in ELISA method than in cultural method showing that it was more sensitive. Moreover, the same serotypes were detected by ELISA showing that no cross reactions were detected between the Mycoplasma species which emphasizes the specificity of ELISA test. ELISA also was proven to be rapid; decreasing the time of diagnosis from 10 days to 3 hours.