Real-time polymerase chain reaction (PCR) is currently used widely for the diagnosis of infectious diseases. We evaluated the time required for which real-time PCR can detect bacteria that had been killed in vitro by different treatments. Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, and Escherichia coli each was treated with alcohol-, antibiotics-, and heat-treatment. Inactivation state of DNA in each sample was confirmed by real-time PCR, including methicillin-resistant Staphylococcus (MRS)-PCR targeting the mecA gene and universal PCR targeting 16S rRNA gene. Each strain was confirmed to be culture-negative after treatment. The time for which MRS-PCR could detect viable MRSA treated by alcohol, vancomycin, linezolid, and heat was confirmed to be less than 16, 8, 12, and 8 weeks, respectively. The time period of universal PCR for detection of viable S. epidermidis treated by alcohol, cefazolin, and heat was less than 4, 20, and 20 weeks, whereas that for viable E. coli were 8, 20, and 4 weeks, respectively. Our studies reveal that the real-time PCR method can detect DNA in killed bacteria for several weeks, but this capability decreases with time and is lost at 20 weeks after treatment.