A brain abscess is a life-threatening infection, frequently with serious sequelae. Culture-based methods present many limitations and do not enable an exhaustive documentation of the bacterial flora. 16S rRNA-based amplification, cloning, and high-throughput sequencing have dramatically increased the number of identified agents of brain abscesses, showing that the causative flora is polymicrobial in up to 40 % of cases, with the presence of at least one anaerobic bacterium. In contrast, real-time polymerase chain reaction (PCR) technology is an appealing alternative to culture-based methods for diagnosing brain abscesses due to its speed, sensitivity, and specificity. Molecular typing is available for several bacterial and fungal genera, and this user-friendly tool is accessible for the clinical microbiology laboratory to diagnose microbes involved in a brain abscess. This article reviews the applications of the currently available tools for the etiological diagnosis of a brain abscess.