ObjectiveTo conduct an epidemiological investigation, isolate and identify the pathogenic bacteria, and perform whole-genome sequencing (WGS) analysis of a food poisoning outbreak in Suzhou High-tech Zone, so as to provide references for the prevention and management of similar incidents. MethodsAn epidemiological investigation was carried out on affected individuals and restaurant staff. Multi-pathogen screening was performed on rectal swab samples from affected cases and restaurant staff, as well as on environmental swab samples from food preparation areas and retained food samples. WGS was performed on the pathogens isolated from the samples. ResultsA total of 15 cases were identified through hospital visit record reviews and on-site inquiries. Of the 15 case samples, 14 tested positive for Staphylococcus aureus (SA). In addition, 3 out of 15 staff samples and 6 out of 34 retained food samples tested positive for SA. WGS results revealed that the 6 food-derived isolates and 13 case-derived isolates shared the same sequence type (ST), Staphylococcal Protein A (SPA) type, and enterotoxin gene profile, with whole-genome single nucleotide polymorphism (wgSNP) differences of ≤5. Two novel SA sequence types: ST9159 and ST9161, were also identified in this study. ConclusionThis food poisoning outbreak is caused by contamination food with SA harbouring the seb and sec enterotoxin genes. The primary causes of contamination and cross-contamination included improper food handling practices, the use of the same utensils for raw and cooked foods, and extended storage of certain dishes at room temperature. Strengthening sanitary supervision in food handling facilities is crucial for reducing the occurrence of similar incidents.