In conclusion, the PMA-PCR assay combined with IAC described in this paper was specific, sensitive, and accurate for the simultaneous detection of C. sakazakii and S. aureus, and B. cereus in spiked infant food samples. The application of IAC effectively eliminated false negative results, while the false positive results were eliminated with PMA treatment before DNA extraction. In addition, the application of an efficient DNA extraction method for the PMAmPCR assay effectively eliminated PCR inhibitors, which resulted in an LOD of 101 CFU/g for C. sakazakii and S. aureus, and 100 CFU/g for B. cereus in spiked infant food samples after 12 h of enrichment even under the interference from non-target bacteria at a high concentration. Thus, the PMA-PCR-IAC assay holds promise as auseful tool for simultaneous detection of C. sakazakii, S. aureus, and B. cereus in infant food products.