© 2022 Elsevier LtdObjective: Although anaerobic bacteria are important agents of a wide variety of serious infections, they are overlooked often in the etiology of infection due to difficulties in isolation and detection. The aim of this study was to develop a new multiplex PCR panel that could detect Bacteroides, Fusobacterium, Prevotella, Veillonella, Clostridium, Peptostreptococcus, and Actinomyces bacteria, which are the most frequently isolated from anaerobic infections, at the genus level. Method: Aerobic and anaerobic cultures were performed on 46 clinical specimens, with suspicion of anaerobic infection and were sent to the laboratory. DNA isolation was performed with the same samples and anaerobic bacteria were detected by the multiplex PCR test developed in the study. Result: The analytical sensitivity of the multiplex PCR assay was found to be 1–103 CFU/ml, depending on the bacterial species. In this study, anaerobic growth was observed in eight (17.4%) of 46 clinical samples. The multiplex PCR test detected 35 anaerobic bacteria from 20 (43.5%) of 46 clinical samples. The most common anaerobes isolated from clinical specimens by the multiplex PCR assay were Prevotella spp. (37.1%) and Fusobacterium spp. (22.9%) while Clostridium spp. (14.3%), Peptostreptococcus spp. (11.4%), Bacteroides spp. (8.6%), and Veillonella spp. (5.7%) followed these genera. Conclusion: As a result, it was concluded that the multiplex PCR panel developed in this study eliminates problems in the detection of anaerobes based on culture, provides more accurate detection of anaerobic bacteria from clinical specimens, takes a shorter time, and allows more accurate infection treatment.