Introduction. As single-celled microorganisms, bacteria are responsible for various diseases and are currently a significant public health concern due to their high resistance to antimicrobials through various mechanisms. Staphylococcus aureus, one of the predominant species in humans, is associated with virulence factors, notably enterotoxins A, B, C, D, and E, which can cause Foodborne Diseases (FBD). Objective. To determine the antimicrobial susceptibility and presence of enterotoxins in Staphylococcus aureus isolated from fresh cheese sold in municipal markets in Cuenca, Ecuador. Methodology. This descriptive, cross-sectional study employs phenotypic and molecular methods to determine antimicrobial susceptibility and identify enterotoxins. Results. Microbiological and molecular biology techniques confirmed the presence of three positive samples for S. aureus using nucA and femB genes. On the other hand, the susceptibility test revealed a high resistance percentage to penicillin in all samples. However, clindamycin, erythromycin, and cefoxitin showed complete sensitivity, so resistance to penicillin was confirmed by amplifying the blaZ gene. No enterotoxins were detected using molecular methods. Conclusion. Sixty-two-point five percent of positive samples showed resistance to penicillin, as confirmed by blaZ gene amplification. Conversely, 100% demonstrated sensitivity to clindamycin, erythromycin, and cefoxitin.