BACKGROUND Intra-abdominal abscesses are localized collections of pus, which generally arise from a breach in the normal mucosal defense barrier that allows bacteria from gastrointestinal tract, and less commonly from the gynecologic or urinary tract, to induce inflammation, resulting in an infection. The microbiology of these abscesses is usually polymicrobial, associated with the primary disease process. However, the microbial identity, diversity and richness in intra-abdominal abscesses have not been well characterized, due in part to the difficulty in cultivating commensal organisms using standard culture-based techniques. METHODS We used culture-independent 16S rRNA Illumina sequencing to characterize bacterial communities in intra-abdominal abscesses collected by percutaneous drainage. A total of 43 abscess samples, including 19 (44.2%) Gram stain and culture-negative specimens, were analyzed and compared with results from conventional microbiologic cultures. RESULTS Microbial composition was determined in 8 of 19 culture-negative samples and 18 of 24 culture-positive samples, identifying a total of 221 bacterial taxa or operational taxonomic units (OTUs) and averaging 13.1 OTUs per sample (interquartile range, 8-16.5 OTUs). Microbial richness for monomicrobial and polymicrobial samples was significantly higher than culture-negative samples (17 and 15.2 OTUs vs 8 OTUs, respectively), with a trend toward a higher microbial diversity (Shannon diversity index of 0.87 and 1.18 vs 0.58, respectively). CONCLUSIONS The bacterial consortia identified by cultures correlated poorly with the microbial composition determined by 16S rRNA sequencing, and in most cases, the cultured isolates were minority constituents of the overall abscess microbiome. Intra-abdominal abscesses were generally polymicrobial with a surprisingly high microbial diversity, but standard culture-based techniques failed to reveal this diversity. These data suggest that molecular-based approaches may be helpful for documenting the presence of bacteria in intra-abdominal abscesses where standard cultures are unrevealing, particularly in the setting of prior antibiotic exposure.