IntroductionRaw milk is a nutrient‐rich food, but it may harbour harmful bacteria, such as enterotoxigenic Staphylococcus aureus (S. aureus), which can cause staphylococcal food poisoning. Antibiotic resistance of S. aureus in raw milk can increase the risk of such infections, particularly among susceptible individuals.ObjectiveThis study aimed to investigate the prevalence of enterotoxin genes a, d, g, i and j and the antibiotic resistance of S. aureus isolated from raw milk samples.MethodsDuring a 6‐month sampling period, 60 raw milk specimens were obtained from diverse locations in Yazd province, Iran. Antibiogram profiling was conducted via the disc diffusion method. In addition, staphylococcal enterotoxin (SE) genes a, d, g, i, and j were detected through real‐time PCR analysis.ResultsBacteriological assays confirmed the presence of S. aureus in 11 samples (18.3%). All isolates demonstrated 100% resistance to penicillin G but exhibited sensitivity to vancomycin, while resistance to other antibiotics ranged from 36.4% to 45.5%. The prevalence of enterotoxin genes in these strains showed variable distribution, with sea being the predominant SE (45.5%), followed by sed (36.4%), seg (18.2), sej and sei (9.1% each).ConclusionsThis study discovered the presence of multiple enterotoxins in S. aureus strains obtained from raw milk samples. These strains also demonstrated resistance to a variety of antibiotics. Since enterotoxigenic S. aureus is known to cause human food poisoning, monitoring food hygiene practices, especially during raw milk production, is critical.