Anthropogenic activities exert significant pressure on aquatic ecosystems, often compromising water quality and public health. This study investigated microbial communities in 4 tributaries of the Bogotá River in Colombia by sampling at 2 points: upstream (near the source) and downstream (near the Bogotá River mouth). The Bogotá River, a critical waterway in Colombia, faces pollution from industrial, agricultural, and urban discharges. Using a combination of traditional physicochemical and microbiological analyses with shotgun metagenomics, we assessed the impact of human activities on water quality and microbial composition. Results revealed diverse microbial communities, including species with bioremediation potential (e.g., Acidovorax sp., Dechloromonas aromatica, and Polaromonas naphthalenivorans), predominantly in upstream samples. By contrast, downstream samples contained a higher abundance of potentially pathogenic species (e.g., Acinetobacter johnsonii and Pseudomonas aeruginosa). Molecular markers indicating health risks were identified, including genes like adeF (associated with fluoroquinolone resistance) and tetO (linked to tetracycline resistance), commonly used in human and veterinary medicine. Metagenome-assembled genomes were reconstructed for Aliarcobacter cryaerophilus, A. johnsonii, and Succinivibrio sp., providing deeper insights into the genetic and functional potential of these species. Our findings highlight the degraded water quality in downstream areas, reflecting the impact of human activities and emphasizing the need for effective water management strategies to restore ecosystem health. This study provides a foundational understanding of microbial diversity in Bogotá River tributaries, essential for future monitoring and mitigation efforts.