Diagnosis of cases of Mycoplasma mastitis is particularly challenging due to their unique biological characteristics, which complicate diagnosis and treatment. Hence, accurate and quick diagnostic tests for early detection of Mycoplasma mastitis are essential to initiate appropriate interventions or culling. The objective of this research is to estimate the diagnostic performance of the molecular microarray assay (MMA) against bacterial culture for the diagnosis of bovine intramammary infections (IMI) with Mycoplasma spp., using a gold standard approach and the Kappa agreement coefficient. A total of 395 quarter milk samples were collected from cows in 31 dairy herds with conventional milking systems in California, USA. Following dairy personnel practices, milk samples were collected from the lactating cows showing abnormal milk characteristics and shipped within 24 hours to the laboratory for bacterial culture and MMA examination. Milk samples with positive growth were confirmed via PCR test to eliminate misdiagnosis of Acholeplasma spp. Eighty-seven cows (22%) were positive for Mycoplasma spp. IMI and the test accuracy was 88.4%. The sensitivity of MMA was 90.8% (95% CI (Confidence Interval): 82.68–95.95), and the specificity was 87.66% (95% CI: 83.46–91.12). The positive predictive value of MMA in these herds was 67.52% (95% CI: 60.51–73.83), and the negative predictive value was 97.12% (95% CI: 94.57–98.49). Calculated Kappa coefficient was 0.70 (95% CI: 0.618–0.778). The high estimates of sensitivity and specificity of MMA suggest its usefulness as a routine and quick test for accurate diagnosis of Mycoplasma spp. IMI in dairy cows. Our findings indicate that MMA holds promise for enhancing the detection of Mycoplasma spp. and could potentially revolutionize diagnostic practices in the dairy industry and supports udder health management.