Background: Although a microbiological diagnosis of pleural infection is clinically important, it is often complicated by prior antibiotic treatment and/or difficulties with culturing some bacterial species. Therefore, we aimed to identify probable causative bacteria in pleural empyema/parapneumonic effusions by combining 16S ribosomal RNA (rRNA) gene amplification and next-generation sequencing (NGS). Methods: Pleural fluids were collected from 19 patients with infectious effusions and nine patients with non-infectious malignant effusions. We analysed DNA extracted from the pleural fluid supernatant by NGS using the Genome Search Toolkit and GenomeSync database, either directly or after PCR amplification of the 16S rRNA gene. Infectious and non-infectious effusions were distinguished by semi-quantitative PCR of the 16S rRNA gene. Results: Only 8 (42%) effusions were culture-positive, however, NGS of the 16S rRNA gene amplicon identified 14 anaerobes and 7 aerobes/facultative anaerobes in all patients, including Streptococcus sp. (n = 6), Fusobacterium sp. (n = 5), Porphyromonas sp. (n = 5), and Prevotella sp. (n = 4), accounting for >10% of the total genomes. The culture and NGS results were discordant for 3 out of 8 patients, all of whom had previously been treated with antibiotics. Total (2 value in semi-quantitative PCR of the 16S rRNA gene) and specific (total bacterial load multiplied by the proportion of primary bacteria in NGS) bacterial loads could efficiently distinguish empyema/parapneumonic effusion from non-infectious effusion. Conclusion: Combining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic effusion and its causal bacteria. ΔCT