A method for the detection and multiple, simultaneous quantification of any combination of Listeria sp., Staphylococcus aureus, Campylobacter jejuni and/or Escherichia coli O157.-H7, in one or more test samples, by multiplex amplification reaction, using real-time polymerase chain reaction (PCR). The steps in the method are: (a) extracting DNA from the test sample or samples; (b) preparing a reaction mixture specific for the pathogens to be detected and quantified, such that the reaction mixture contains the necessary reagents for enzymatic amplification of the extracted DNA and identification of the pathogens to be detected and quantified; (c) amplifying the reaction mixture by means of multiplex amplification reaction using PCR; and (d) simultaneously determining the presence or absence of and quantification of the pathogens in the test sample or samples. The method has the special feature that (i) the reaction mixture for enzymatic amplification of the DNA has sets of pairs of oligonucleotide primers identified as SEQ ID No. 1 and SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 8, and SEQ ID No. 10 and SEQ ID No. 11, and probes with oligonucleotide sequences identified as SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9 and SEQ ID No. 12; (ii) the presence or absence of and quantification of said pathogens in any combination is determined by means of a fluorescent signal or fluorescence emission specific for each pathogen.