Background: The research project in Lahore, Pakistan, seeks to detect S. aureus in drinking water, raw milk, and yogurt samples due to health concerns. S. aureus is a pathogenic bacterium with potential risks if present in food and water sources.Method: In this study, 300 samples of raw milk, yogurt, and drinking water were collected in Lahore. The presence of S. aureus was determined through morphological, microscopic, and biochemical methods. The biochemical analysis included testing for specific features of S. aureus. Disk diffusion technology was employed to assess the antimicrobial susceptibility of the isolates, following the recommendations of the Clinical Laboratory Standards Institute. Molecular confirmation was achieved through 16S rRNA sequence analysis using universal and specific primers.Results: The investigation uncovered that 6% of drinking water samples, 9% of yogurt samples and 58% of raw milk samples were tainted with S. aureus. These findings were further validated through 16S rRNA sequence analysis, affirming their reliability. S. aureus exhibited notable resistance rates, with 100% resistance to penicillin and 95% resistance to erythromycin. Conversely, resistance to ciprofloxacin and gentamicin was lower, at 10% and 5% each, indicating the potential efficacy of these antibiotics in treating S. aureus infections.Conclusion: The study emphasizes the risk of S. aureus infection from raw milk consumption in Lahore, Pakistan, due to inadequate sanitary practices. It stresses the necessity of implementing stricter measures in dairy production and water treatment to ensure public safety and reduce multidrug-resistant bacteria prevalence. Continuous monitoring and preventive actions are vital for safeguarding public health.Keywords: S. aureus; Antibiotic resistance; Food and water; Lahore, Pakistan; Sanitary measures