The current study aimed to determine the prevalence of Mycoplasma bovis in cattle in Nineveh Governorate, Iraq, using the culture method, indirect enzyme-linked immunosorbent assay (i-ELISA), and conventional polymerase chain reaction (c-PCR) technique, to evaluate the efficiency of the various laboratory methods used in this study. During the period from March 2022 to February 2023, a total of 352 nasal swabs were collected randomly from cattle and tested using the culture method and conventional polymerase chain reaction (c-PCR) technique (amplifying the uvrC gene) for isolation and molecular detection of M. bovis , respectively. Moreover, 352 blood samples were collected from the same animals for the detection of antibodies against M. bovis  using i-ELISA. The results observed that the prevalence of M. bovis was 23.57% (83 out of 352) using the culture method, 30.68% (108 out of 352) using the c-PCR technique, and 47.15% (166 out of 352) using i-ELISA. According to the Kappa value (0.807), there was perfect agreement between the c-PCR technique and the culture method, with sensitivity, specificity, and accuracy were 98.79%, 90.33%, and 92.32% for c-PCR technique respectively. While there was moderate agreement between i-ELISA test and the culture method based on Kappa (0.444), with sensitivity, specificity and accuracy were 89.15%, 65.79%, and 71.30% for i-ELISA, respectively. This study was concluded that M. bovis  is prevalent in Nineveh Governorate and the PCR technique is the more efficient tool than ELISA for detection of M. bovis in suspected samples.