The presence of Staphylococcus spp. in milk and milk products is a concern for dairy farmers, industry and government agencies responsible for public health. Because these microorganisms are important pathogens of mastitis, the udder of the infected animal is a major source of contamination of raw milk. S. aureus is the species most frequently associated with mastitis and outbreaks or cases of food poisoning. Other species of Staphylococcus have also been associated, but often much less, to staphylococcal food poisoning. The objective of this project was to identify and evaluate the enterotoxigenic potential of 500 Staphylococcus spp. isolated from mastitis, refrigerated raw milk, pasteurized milk, Minas frescal cheese and Coalho cheese and carry out the molecular characterization of coagulase positive Staphylococcus and enterotoxigenic strains. Staphylococcus coagulase positive and enterotoxigenic strains were identified by the PCR technique using specific primers. Staphylococcus coagulase negative confirmed as enterotoxigenic by PCR were identified using the API®-STAPH system (BioMérieux, Marcy-l'Etoile - France). The isolates had their electrophoretic profile determined by pulsed field electrophoresis (PFGE) and 10% of enterotoxigenic ones had the PCR fragments sequenced for each gene. Staphylococcus presenting classical enterotoxin genes was evaluated in vitro for enterotoxin production by using commercial kits. PCR procedures were established for the rapid identification of coagulase-positive Staphylococcus-causing mastitis and enterotoxigenic strains, and a better knowledge of the mastitis and milk-borne enterotoxigenic Staphylococcus. These results could help improving the productive sector and public health agencies in adopting measures or programs to control these microorganisms in dairy herds and to ensure the production of safer dairy products.