Staphylococcus aureus (S. aureus) is responsible for most foodborne outbreaks. This study aimed to evaluate the prevalence of S. aureus in the food chain, humans, and environment in Mansoura to evaluate one health aspect. A total of 210 samples from raw milk, meat (minced meat, meat by products, and processed meat products), and poultry meat were randomly selected (seventy samples from each); in addition, twenty samples were taken from their surrounding environment and the hands of their workers. These samples were collected from small markets, and student residences located in Mansoura, Egypt, during the period of September 2020 and March 2021. For S. aureus isolation, samples were subjected to standard culture techniques. Identification of S. aureus colonies was performed by molecular confirmation using PCR for the biochemically suspected S. aureus isolates by utilizing a molecular marker that targeted the species-specific thermonuclease-nuc gene. Out of 230 samples, 50 isolates were determined to be S. aureus. The overall prevalence of S. aureus isolates in our study was 21.7% (50/230). S. aureus was present in 35.7% (25/70) of the raw milk sampled. S. aureus isolates were found in 13.3% (4/30), 10% (2/20), and 15% (3/20) of the meat, meat by-products, and processed meat products, respectively. Fourteen (20%) of the seventy samples of chicken meat contained isolates of S. aureus. Ten percent (1/10) of the worker's hand swabs and samples from nearby surfaces contained S. aureus isolates. To improve one health aspect, tracking food and monitoring farm animals must be built.