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Staphylococcus aureus is one of the major food contaminants worldwide, and its enterotoxins are documented as food poisoning and bioterrorism agents. In the present study, an attempt was made to account on the incidences of toxigenic S. aureus and its antibiotic resistance profiles in ready to eat bakery food products from different parts of Southern India (Andhra Pradesh, Karnataka, Kerala, Tamil Nadu, and Telangana). A total of 100 food samples, including milk, cake, cheese and chicken products were assessed for S. aureus and Staphylococcal Enterotoxin B (SEB) by PCR. Among the subjected food samples, a total of 51 isolates belong to genus Staphylococcus and out of that, 34 isolates were S. aureus . Among 34 S. aureus isolates, 14 isolates were found positive for SEB. The PCR results were further co-evaluated with in-house developed aptamer linked immunosorbent assay (ALISA) for the specific and sensitive detection of SEB. The obtained ALISA results were promising and found consistent with PCR analysis. Furthermore, 24%, 47%, 91%, 82%, 59%, and 47% of S. aureus isolates were found resistant to chloramphenicol, methicillin, penicillin, ampicillin, erythromycin, and oxacillin, respectively and concluded as a multidrug resistance (MDR). In conclusion, the present study revealed high presence of toxigenic and MDR resistant S. aureus species among the studied regions of Southern India. The present study cautions the need of stringent food safety regulations in India to control the toxigenic and MDR S. aureus from food sources and to minimize the risks associated with S. aureus .