Millions of cases are reported every year, globally, on foodborne diseases that pose a significant hazard to public health. Primarily caused by the pathogens affecting the gastrointestinal tract and sometimes associated with human waste being used as fertilizers in agriculture, these are generally known as foodborne diseases. The bacterial pathogens include Staphylococcus aureus (S. aureus) which is unique due to its involvement in the production of heat-stable enterotoxins that facilitate the occurrence of severe community- and hospital-acquired infections like food poisoning. Enzyme-linked immunosorbent assay (ELISA) has become an important tool for sensitive and specific detection of bacterial toxins originating from Staphylococcus aureus and other toxigenic bacteria including their presence in complex matrices such as food. The chapter presents a study on principles and applications of ELISA to detect gram-positive bacterial toxins causing foodborne diseases including those produced by S. aureus as model products. In conclusion sandwich ELISA using capture and detector antibodies is recommended for toxin identification and quantification in foods. ELISA tests allow rapid screening for toxins to prevent outbreaks or implement safety measures at the public health level mainly through careful sample preparation before use on this test technique.