OBJECTIVE To report the potential usefulness of multiplex polymerase chain reaction (mPCR) for diagnosing superinfection keratitis caused by herpes simplex virus-1 (HSV-1), bacteria and fungus. METHODS Case series. Corneal scrapings were analyzed with mPCR for human herpes virus 1-8, bacterial 16S ribosomal DNA (rDNA) and fungal 28S rDNA. RESULTS Case 1 was a 69-year-old man who presented with refractory infectious keratitis. PCR examination was positive for bacterial 16S rDNA and negative for fungal 28S rDNA. HSV-1 was not examined at this time. A geographic ulcer arose after 2 months of intensive antibacterial treatment. Herpes simplex keratitis (HSK) was suspected; PCR analysis was positive for HSV-1. Corneal scrapings obtained at the initial visit were re-analyzed and found to be HSV-1 positive. Thus, it turned out that this was a case of superinfection keratitis caused by bacteria and HSV-1. Case 2 was a 60-year-old man with corneal ulcer who had received unsuccessful treatment with antibiotics. mPCR analysis was positive for HSV-1, bacterial 16S rDNA and fungal 28S rDNA. The patient was diagnosed with superinfection keratitis caused by HSV-1, bacteria and fungus. Case 3 was an 82-year-old woman who had been treated for HSK and then developed bacterial keratitis during treatment. mPCR analysis was positive for HSV-1 and bacterial 16S rDNA. The patient was diagnosed with superinfection keratitis caused by HSV-1 and bacteria. CONCLUSIONS Superinfection keratitis is hard to diagnose because of its atypical manifestation. mPCR has the potential to allow prompt diagnosis and appropriate treatment in these cases.