This study was designed to identify the bacteria and yeasts from the milk samples of dairy cattle presenting with subclinical mastitis and evaluate their antimicrobial susceptibility. We collected a total of 52 milk samples from cows across three farms in San Salvador El Seco (Puebla, Mexico). Microbial isolation was performed using microbiological techniques followed by taxonomic identification of bacteria and yeasts. Antimicrobial susceptibility was evaluated using the guidelines provided by the Clinical Laboratory Standard Institute (CLSI). 1 We identified three genera and six species of yeasts including Candida glabrata, C. krusei, C. lipolytica, Cryptococcus laurentii, Rhodotorula rubra, and R. glutinis and five species of bacteria, including Staphylococcus saprophyticus, S. aureus, S. hominis, S. epidermidis, and Streptococcus disgalactiae. All of the yeast strains were sensitive to amphotericin B; 1/23 (4.3%) were resistant to ketoconazole and nystatin, 10/23 (43%) were resistant to fluconazole, and 13/23 (53%) were resistant to 5-fluorocytosine. The dominant genus isolated was Candida, with the most abundant groups being C glabrata and C. krusei. Resistance to 5-fluorocytosine was observed in all yeasts except C. lipolytica, while both S. aureus and S. epidermidis were resistant to oxacillin and dicloxacillin. S. hominis was resistant to gentamicin. These antimicrobials are still used in bovine therapy for mastitis, directly affecting healthy cattle and, therefore, raw milk.