To evaluate a nested PCR protocol for Listeria monocytogenes detection, Minas Frescal cow's milk cheeses were produced, artificially inoculated with this pathogen at concentrations ranging from 1 to 1,000 CFU/g, and stored at 4°C for 10 days. The International Organization for Standardization (ISO) standardized method 11290-1/A1 was used to detect L. monocytogenes in the inoculated samples, and DNA was extracted from aliquots (1, 5, and 10 ml) of 1:10 dilution, followed by a nested PCR protocol for the hlyA gene. The ISO standardized reference method and nested PCR both detected L. monocytogenes at all concentrations and during all storage periods; McNemar's test showed no significant difference (P > 0.05). The results indicated that the nested PCR protocol can be used as a screening test to detect L. monocytogenes in Minas Frescal cheese, allowing earlier detection of the pathogen that can later be confirmed by the ISO standardized reference method.