Food samples containing Staphylococcus aureus pose a serious health risk. This study aimed to examine the prevalence of S. aureus strains in various food samples sourced from Mailsi and Multan. Many food samples including yogurt, bakery products, and raw and cooked food were examined for S. aureus. The isolated strains were confirmed through Mannitol agar fermentation, catalase, coagulase, and urease tests. Hemolysis on blood agar and biofilm formation were also assessed to determine toxin production. Antibiotic sensitivity testing was conducted using the Kirby Bauer method on MH agar, and multiple antibiotics were tested. Out of all samples, a total of 50 S. aureus strains were obtained, mainly from milk and milk-based products. Yellow colonies on mannitol salt agar confirmed S. aureus presence, with all isolates testing positive for coagulase, catalase, and urease. The presence of hemolysins: beta, gamma, and alpha were revealed by hemolysis assays. Biofilm assay results showed variation among the strains, with some categorized as strong, moderate, or weak biofilm formers. Regarding antibiotic sensitivity, most strains exhibited multidrug resistance, particularly against certain antibiotics. Vancomycin showed varying susceptibility patterns, some strains showed susceptibility and intermediate resistance, whereas only milk samples showed resistance. This study emphasizes the prevalence of MDR S. aureus strains in food samples. The study underscores the significance of antimicrobial stewardship programs and stringent food safety measures in preventing the spread of antibiotic-resistant strains and reducing foodborne illnesses. Further research is needed to explore the mechanisms behind antibiotic resistance and toxin production in S. aureus strains from food samples.