Mastitis still remains an economic disease in dairy farms all over the world. This study aimed to report the prevalence of this disease in large-scale farms in Vietnam, the pathogens profile, and first report the detection of Mycoplasma bovis (M. bovis) by applying conventional culture and real-time PCR (polymerase chain reaction) methods. We examined 64,802 enrolled dairy cows from six farms in Vietnam, yielding 1,874 (2.9%) total cases of clinical mastitis. The majority of the 14 pathogens were identified. Klebsiella spp. accounted for 21.0% of the total isolates. Streptococcus uberis (S. uberis), Escherichia coli (E. coli), other Streptococcus spp, and Coagulase-Negative Staphylococci (CNS), followed at 12.6, 7.6, 6.0, and 5.5 percent, respectively. The rest pathogens include Pseudomonas spp., Enterococcus faecalis, Enterobacter spp., Bacillus spp., Citrobacter spp., Pasteurella spp., Proteus spp., and Staphylococcus aureus were isolated with less than 1% each. To the best of our knowledge, this is the first report for detecting M. bovis in mastitis milk samples in Vietnam, where 35 out of 1,422 samples submitted in 2022 were M. bovis positive. Moreover, of the 35 positive cases, 11 cases had only M. bovis, while 24 cases had M. bovis and other pathogens, including 13 Enterobacteriaceae, 7 Streptococcus spp., and 4 CNS.