Mycoplasma bovis is an important pathogen associated with several clinical diseases in cattle, such as mastitis, arthritis, and pneumonia. TableIn this study, we aimed to identify miRNA candidate biomarkers associated with inflammation in Mycoplasma bovis-infected milk samples and normal milk samples of Holstein-Friesian (HF) and Doğu Anadolu Kırmızısı (DAK) cows in Turkey. The expression levels of miRNAs in milk from mastitis-infected cows and uninfected cows were analyzed using a qRT-PCR. The results revealed that miR-21, miR-146a, miR-155, miR-222, miR-383, miR-200a, miR-205, miR-122, and miR-182 were upregulated in mastitis milk. Among the miRNA candidate biomarkers, miR-21 and miR-222 were significantly upregulated only in mastitis milk samples from HF cows, and miR-146a and miR-383 were significantly upregulated only in mastitis milk samples from DAK cows. These results shed light on miRNA candidate biomarkers in milk from HF and DAK cows with subclinical mastitis. The upregulated miRNAs detected in the present study could be used as biomarkers in the diagnosis of subclinical mastitis caused by Mycoplasma bovis.