This study aims to develop a rapid detection method based on multiplex real-time quantitative PCR technology for accurate identification and quantification of common pathogenic bacteria on face masks, to assess the hygienic condition of masks and control nosocomial infections. Methods Using TaqMan probe-based real-time fluorescence quantitative PCR as the core technology, specific primers and fluorescently labeled probes were designed and optimized for simultaneous quantification of specific gene sequences from three pathogens through one-tube multiplex detection. The experiment began by screening specific target genes and sequences from the NCBI database and designing primers and probes. Subsequently, quality testing of primers and probes was conducted, and standard samples were prepared through amplification of specific sequences and TA cloning. Finally, standard curves were established, and mask samples were tested. Results After optimizing experimental conditions, an efficient and stable multiplex real-time quantitative PCR detection system was successfully established. The detection efficiency of standard samples for three pathogens was evaluated at different concentration gradients. The detection data showed that the primers and probes had high specificity for target pathogens, and the amplification efficiency and signal intensity met experimental requirements. In the detection of mask samples, the method accurately quantified the genomic DNA copy numbers of Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus hemolyticus, thereby assessing the degree of microbial contamination on masks. Conclusion The rapid detection method for pathogenic bacteria on masks based on multiplex real-time quantitative PCR technology demonstrates high sensitivity, specificity, and speed, providing strong technical support for clinical and public health