Headwater streams are crucial for the health and resilience of entire watersheds; therefore, comprehending how human activities disrupt the structure and function of these vulnerable ecosystems is paramount. Bacterial assemblages respond to a myriad of environmental changes and have the potential to indicate subtle anthropogenic impacts to relatively pristine headwater streams. We collected benthic samples from 46 headwater streams throughout the upper Delaware River Basin, USA. Streams were relatively pristine with upstream forest covers >50%. Bacterial community compositions for each stream were characterized following DNA extraction, amplification of the 16S rRNA V4 gene, and sequencing in the laboratory of Mehrdad Hajibabaeui at the University of Guelph. Nonmetric multi-dimensional scaling was used to visualize between-site differences and potential relationships with environmental variables. We found that bacterial community composition was most strongly related to canopy cover. It is likely that increased algal dominance of stream biofilms associated with reduced riparian canopy leads to detectable shifts in the bacterial community. These results demonstrate how subtle impacts, such as a reduction in the riparian tree canopy, can have measurable effects on the microbial assemblages.