The rapid detection of etiological agents is important for the successful treatment of iliopsoas abscess (IPA). The purpose of this study was to investigate the clinical utility of a real-time polymerase chain reaction (PCR) that targets the mecA gene for methicillin-resistant staphylococci (MRS) and the 16S rRNA gene for pan-bacteria. Our retrospective diagnostic study included 22 patients exhibiting IPAs and four patients with noninfectious iliopsoas mass regions who underwent computerized tomography or ultrasonography-guided biopsy and/or surgical treatment. Clinical symptoms, serum data, imaging analysis, and tissue microbiological culture were utilized for the diagnosis of IPA. The diagnostic accuracy of real-time PCR was determined based on the diagnosis of IPA and microbiological culture results. The microbiological culture was positive for 12 IPA cases that included 2 MRSA infections. Among 12 culture-positive IPA cases, 16S rRNA-PCR was positive in 12 and MRS-PCR in two. Among 10 culture-negative IPA cases, including 3 TB cases, 16S rRNA-PCR was positive in 8 and MRS-PCR in 2. In noninfectious iliopsoas mass patients, neither 16S rRNA nor MRS-PCR detected bacterial DNA. The sensitivity, specificity, positive predictive, and negative predictive values of 16S rRNA-PCR for diagnosing IPA were 0.91, 1.00, 1.00, and 0.67, respectively, while those for the diagnosis of MRS infection with MRS-PCR were 1.00, 0.92, 1.00, and 0.50, respectively. Real-time PCR targeting bacterial DNA can detect bacterial DNA in culture-negative cases and offer improved detectability of MRS infection in IPA patients.