An increase in polycyclic aromatic hydrocarbon (PAH) pollution poses significant challenges to human and ecosystem health in the Three Gorges Reservoir (TGR) of the Yangtze River. Based on the combination of PAH analysis with qPCR and high-throughput sequencing of bacteria, 32 topsoil samples collected from 16 sites along the TGR were used to investigate the distribution and biodegradation pathways of PAHs in the water-level-fluctuation zone (WLFZ). The results indicated that the concentrations of PAHs were 43.8–228.2 and 30.8–206.3 ng/g soil (dry weight) under the high- and low-water-level (HWL and LWL) conditions, respectively. The PAH concentration in urban areas was higher than that in rural areas. Under both the HWL and LWL conditions, the abundance of the bamA gene, a biomarker of anaerobic PAH biodegradation, was significantly higher than that of the ring-hydroxylating-dioxygenase (RHD) gene, a biomarker of aerobic PAH biodegradation. The abundance of the bamA gene was significantly positively correlated with PAHs (R² = 0.8), and the biodegradation percentage of PAHs incubated anaerobically was greater than that in the aerobically incubated microcosm experiments. These data implicated a key role of the anaerobic pathway in PAH biodegradation. Co-occurrence network analysis suggested that anaerobic Anaerolineaceae, Dechloromonas, Bacteroidetes_vadin HA17 and Geobacter were key participants in the biodegradation of PAHs. The diversity analysis of functional bacteria based on the bamA gene and microcosm experiments further demonstrated that nitrate was the primary electron acceptor for PAH biodegradation. These findings provide a new perspective on the mechanism of PAH biodegradation in the TGR and knowledge that can be used to develop strategies for environmental management.