Benthic ecosystem of river sediment, composing of bacterial, fungi, algae, protozoan, metazoan communities, and bottom-dwelling fish, is a complex biological community across multiple trophic levels. Yet few study has attempted to evaluate the potential effects of polycyclic aromatic hydrocarbons (PAHs) contamination on the biodiversity of the whole sedimentary communities. In this study, Beiluo river located in the Loess Plateau (China) was used as a representative site to reveal the river sediment community shifts and potential interaction with PAHs. We hypothesize that low molecular weight (LMW) PAHs in Beiluo River was the primary stressor for the abundance and diversity loss for benthic communities. The objectives of this study were: 1) to investigate the distribution of multitrophic communities in sediments using DNA metabarcoding, including 16S-, 18S-, COI- and 12S rRNA sequencing for bacteria, microbial eukaryotes, metazoan, and fish, respectively; 2) to reveal the spatial distribution levels of PAHs and source apportionment; 3. to unravel whether potential PAHs in watershed sediments links to the shifts in community structure and diversity. Here, the concentrations of PAHs in sediments ranged from 25.95 to 1141.35 ng/g. Furthermore, the high concentrations of MMW PAHs (ANT, PHE), mainly deriving from coal and biomass combustion, were negatively linked with the Shannon’s diversity index and richness of decomposer-bacteria (Proteobacteria), primary consumer-protists (Apicomplesa) and secondary consumer-fish (Pleuronec). High molecular weight (HMW) was linked to the reduced diversity and richness of algae (Chlorophyta, Chrysophyceae) and fish (Cichliformes), though the occurrence level of HMW was low. Surprisingly, no evident connection on the biological community was observed from the low molecular weight (LMW) PAHs deriving from the oil spill. In conclusion, MMW and HMW PAHs derived from coal and biomass combustion and vehicle exhaust in river sediments may reduce the river sedimentary biodiversity of primary producers and secondary consumers.