Bovine mastitis caused by Mycoplasma bovis represents a major problem for dairy industry all over the world. The present study was designed to determine the occurrence of Mycoplasma bovis in mastitis milk from different dairy herds in Egypt, and determination of the most appropriate diagnostic protocol for rapid, accurate diagnosis. That will allow a quicker segregation and culling of infected animals and lower morbidity rates as well, and leading to reduction in economic losses. Mastitis milk samples (n=249) were collected and examined for Mycoplasma isolation using conventional cultural method then identified by Mycoplasma bovis specific PCR using mb-mp 81 gene primers. Among the 249 examined milk samples, 18 samples (7.23%) were positive for Mycoplasma bovis. For comparison of various diagnostic methods for detection of Mycoplasma bovis, twenty-two milk samples were selected randomly from the collected samples and analyzed by three methods, PCR directly from milk, Culture followed by PCR, and the antigenic ELISA. The finding of the comparative study between the three used techniques revealed that Culture followed by PCR 9/22 (40.9% Positive results) was the most sensitive methods for detection of Mycoplasma bovis in milk samples followed by AgELISA 7/22 (31.82% Positive results) and PCR directly from milk 2/22(9.09% positive results).