Aquatic environments across Canada constantly receive anthropogenic discharge. In this context, water is considered the main vehicle for the dissemination of pathogens into the environment. The current assessment of microbiological water quality indicators focuses on fecal bacteria such as E. coli, overlooking viruses and protozoa. Monitoring impacted freshwater ecosystems without focusing on single microorganisms offers fundamental data to develop new microbial indicators for aquatic contamination. A longitudinal, 8-month amplicon-based analysis was performed in the Assiniboine River to identify the microbial composition, relative abundance, and environmental structuring factors in water associated with different anthropogenic activities. Bacteriophages, bacteria, and microeukaryotes were studied using the major capsid protein gene g23, 16SrRNA, and 18SrRNA genetic markers with Illumina technology. Our analysis revealed the riverine ecosystems were compounded in the majority by cyanophages (62%) (Synechococcus phages, and Bellamyvirus genus phages), Escherichia coli phages (26%), Emdodecavirus genus phages (8%), and Slopekvirus genus phages (2%). The most relatively abundant bacterial families across waterways included Sporichthyaceae (20) %, Pseudomonadaceae (19%), Enterobacteriaceae (14%), Burkholderiaceae (8%), Spirosomaceae (7%) , Flavobacteriaceae (3%), Aeromonadaceae (3%), Micrococcaceae (3%) and Nostocaceae (3%). The microeukaryotic structure consisted of Mediophyceae (24%), Cryptomonadales (14%), Choreotrichia (14%), Oligotrichia (3%), Sphaeropleales (7%), Chlamydomonadales (1%), Kappamyceteceae (5%), and Thoracosphaeraceae (3%). The effects of location and sampling months for bacteriophages were inconclusive. Sampling months affected the community structure of bacteria and microeukaryotes significantly. Forested, urban and agriculturalD activities significantly shaped microeukarytic communities. Microbial communities were highly impacted by environmental factors, especially daylight length, water temperature, dissolved oxygen (DO), and precipitation. Our results also revealed complex networks of trophically interacting microorganisms between primary producers, heterotrophic bacteria, and predators (i.e. Oligotrichia). These results are critical to better understanding the dynamic interplay and the effect of temporal changes in microbial fractions as well as providing essential information for new potential indicators for aquatic contamination.