Brain abscesses require prolonged treatment and can involve mixed infections. Detecting the causative organism is crucial while deciding treatment. We describe the case of a 13-year-old healthy boy admitted to our hospital because of difficulty in speech and movement. Computed tomography revealed a subdural abscess spread from chronic sinusitis; endoscopic sinus surgery was performed on the day ofadmission. As the patient’s neurological symptoms did not improve and the abscesscontinued to worsen, urgent burr hole drainage was performed on day 4 of admission; however, the abscess did not improve, and a second burr hole drainage was performed on day 14 after admission. Blood culture before obtained prior to treatment initiation identified methicillin-sensitive Staphylococcus aureus (MSSA), while Streptococcus intermedius was exclusively isolated from the abscess cultures.To investigate the involvement of MSSA in the subdural abscesses, we performed polymerase chain reaction test and 16S rRNA gene analysis. Therefore, MSSA and S. intermedius and Fusobacterium nucleatum have also been detected in the pus of a subdural abscess. In addition, a zone edge test was performed for MSSA, and ampicillin treatment was continued for 6 weeks following the second burr hole drainage because F. nucleatum was not detected in the anaerobic culture. Thereafter,the patient’s neurological symptoms significantly improved; the patient wasdischarged on the 58th day after admission. This case highlights the limitations of relying solely on culture tests for pathogen detection, especially with prior antimicrobial therapy. In the presence of prior administration of antimicrobial agents and differences in culture tests, using bacterial gene detection method may facilitate precise diagnosis and treatment.