Mastitis is the most common disease affecting dairy farms, causing inflammation of the mammary glands and reducing milk quality and production. Incorrect use of antibiotics leads to serious problems in veterinary medicine due to the emergence of multidrug resistant bacteria. The antibiotic resistant bacteria are considered the big problems worldwide. This study aims to isolate pathogenic bacteria from raw milk samples taken from subclinical mastitic cows. In addition, estimation of the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of Zinc oxide (ZnO) nanoparticles was also done against Staphylococcus aureus and Escherichia coli isolated from subclinical mastitic cows. Out of 28 milk samples, 85.7% samples showed positive bacterial isolation while 33.3% samples had single isolates while, 66.7% showed mixed isolates and 4 samples showed negative bacterial isolation. The obtained results showed that the resistance ratio of S. aureus and E. coli to different antibiotics using the disc diffusion technique was highest for Amoxicillin, then Tetracycline, and finally Nalidixic acid. Results of the MIC of ZnO nanoparticles against isolated E. coli and S. aureus were 31.3 µg/mL and 7.8 µg/mL, respectively. While the MBC was 62.5 µg/mL for E. coli and 15.6 µg/mL for S. aureus. It was found that ZnO nanoparticles could be used instead of common antibiotics to treat subclinical mastitis in cows caused by E. coli and S. aureus and to overcome the problems of multidrug resistant bacteria.