Along with rapid urbanization and intensive human activities, polycyclic aromatic hydrocarbon (PAH) pollution in the Pearl River Estuary (PRE) and its effects on the microbial community have attracted extensive attention. However, the potential and mechanism of microbial degradation of PAHs across water and sediment habitats remain obscure. Herein, the estuarine microbial community structure, function, assembly process and co-occurrence patterns impacted by PAHs were comprehensively analyzed using environmental DNA-based approaches. The contamination and distribution of PAHs were jointly affected by anthropogenic and natural factors. Some of the keystone taxa were identified as PAH-degrading bacteria (i.e., genera Defluviimonas, Mycobacterium, families 67–14, Rhodobacteraceae, Microbacteriaceae and order Gaiellales in water) or biomarkers (i.e., Gaiellales in sediment) that were significantly correlated with PAH levels. The proportion of deterministic process in the high PAH-polluted water (76%) was much higher than that in the low pollution area (7%), confirming the significant effect of PAHs on the microbial community assembly. In sediment, the communities with high phylogenetic diversity demonstrated a great extent of niche differentiation, exhibited a stronger response to environmental variables and were strongly influenced by deterministic processes (40%). Overall, deterministic and stochastic processes are closely related to the distribution and mass transfer of pollutants, and substantially affect the biological aggregation and interspecies interaction within communities in the habitats.