Microbial biofilms colonizing streambeds critically drive ecological and biogeochemical processes in streams globally. While their significance is increasingly recognized, the role of bacteriophages (phages) – viruses that infect bacteria – remains underexplored. Here, we introduce the Alpine Lotic Phage (ALP) collection; a pioneering initiative to isolate and characterize phages from alpine streams. We collected over 120 litres of water from a Swiss mountain stream which were filtered, concentrated, and screened against 40 bacterial species isolated from stream biofilms. This effort yielded 60 double-purified phage isolates infecting 15 different bacterial species. Soft-agar assays and electron microscopy revealed diverse plaque morphologies and tailed virus structures, while genome sequencing uncovered a wide range of genome sizes (~27 to 360 kbp). Genomic annotation also indicated the presence of putative temperate phages and numerous uncharacterized genes. We next challenged biofilm-forming bacteria with ALP phages and report that while static biofilms were largely unaffected, phages strongly inhibited biofilm growth under flow conditions in microfluidic devices, suggesting a context-dependent impact of alpine phages on biofilm development. Collectively, the ALP collection highlights an untapped reservoir of phages in alpine streams with efforts ongoing to further expand the viral collection and to explore phage–biofilm interactions in lotic ecosystems.