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Brain abscess is a severe infectious disease with high mortality and mobility. Although culture-based techniques have been widely used for the investigation of microbial composition of brain abscess, these approaches are inherent biased. Recent studies using 16S ribosomal sequencing approaches revealed high complexity of the bacterial community involved in brain abscess but fail to detect fungal and viral composition. In the study, both culture-independent nanopore metagenomic sequencing and culture-based whole-genome sequencing using both the Illumina and the Nanopore platforms were conducted to investigate the microbial composition and genomic characterization in brain abscess. Culture-independent metagenomic sequencing revealed not only a larger taxonomic diversity of bacteria but also the presence of fungi and virus communities. The culture-based whole-genome sequencing identified a novel species in Prevotella and reconstructs a Streptococcus constellatus with a high GC-skew genome. Antibiotic-resistance genes CfxA and ErmF associated with resistance to penicillin and clindamycin were also identified in culture-based and culture-free sequencing. This study implies current understanding of brain abscess need to consider the broader diversity of microorganisms. [ABSTRACT FROM AUTHOR]