Microbes are of critical importance but are a poorly understood component of arctic stream ecosystems. They are responsible for recycling organic matter and regenerating nutrients that are essential to the food webs of aquatic ecosystems. We tested the hypothesis that differences in highly contrasting parent lithologies (non-carbonate and ultramafic), stream habitat (sediments and rocks), and stream biogeochemistry influence the structure of bacterial biofilm communities in arctic streams. Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequencing were used to explore the genetic diversity of bacterial communities in biofilms on sediments (epipssamon) and rocks (epilithon) in stream reaches that drain watersheds with contrasting lithologies in the Noatak National Preserve, Alaska. We observed different patterns in bacterial community composition primarily by stream habitat and secondarily by lithology. Non-metric multidimensional scaling (NMS) ordination of terminal restriction fragment peaks showed highly significant separation between the two habitats and moderately significant separation between lithologies. Positive correlations were detected between particular biogeochemical variables (e.g. nutrients, base cations, and dissolved organic carbon) and bacterial community structure unique to each lithology. Although clone libraries indicated high diversity of bacterial operational units within and across stream sites, biogeographical patterns were observed that depended on lithology type and stream habitat. Our results show relationships at macro- and micro-scales at the landscape level and in ecological niches within a single stream.