Staphylococcus aureus (S. aureus) is the most important bacterial cause of subclinical mastitis (SCM), which is resistant to treatment. This study was conducted on cows with SCM to estimate hematobiochemical changes. It also aimed to isolate and identify multidrug-resistant S.aureus and its antibiotic susceptibility pattern to detect antibiotic-resistant genes. One hundred cattle from different farmholders were examined by the California mastitis test, which revealed that 32 (32%) SCM cases were positive. Blood and milk samples were collected from subclinical cases and 10 control healthy cows.  This study showed a significant increase in AST, ALT, ALP, total leucocytic count, neutrophil, basophil percentage, and CRP, while a significant decrease in the erythrocytic count, total protein and albumin in subclinical cases, compared to the control group. The antibiograms of 32 S. aureus isolates showed high resistance toward penicillin (65.6%), ampicillin (56%), imipenem, and amoxicillin (44%) each. These were followed by tetracycline (40.6%), vancomycin (37.5%), amoxicillin-clavulanic acid, ciprofloxacin (34%) each, and chloramphenicol (31%). All the isolates resistant to the antibiotics encode blaZ, tetK, vanA, optrA, norA, mphC, and mecA genes. The haemato-biochemical parameters associated with SCM can be used as vital indicators of SCM. S. aureus is an important cause of SCM, which is highly resistant to antibiotic treatment. Antibiotic susceptibility tests improve the selection of appropriate antimicrobial drugs and avoid antibiotic resistance. Molecular surveillance is essential for the detection of S. aureus resistance genes.