Urban water bodies serve as critical reservoirs of microbial diversity, with major implications for public health and environmental quality. This study aimed to characterize the microbial diversity of surface waters and wastewater from the Pasto River in the Colombian Andean Highlands, offering insights that may support water quality monitoring efforts. Sampling was conducted at three river sites and one wastewater location. Standard physicochemical and microbiological analyses were performed, including real-time PCR to detect protozoan pathogens Giardia spp. and Cryptosporidium spp. Metagenomic sequencing provided an in-depth taxonomic and functional profile of microbial communities through two complementary approaches: (i) read-based analysis to identify abundant families and species, both pathogenic and beneficial, and (ii) detection of health-related molecular markers, including antimicrobial resistance markers and virulence factors. Physicochemical analyses showed distinct profiles between wastewater and surface water, with wastewater exhibiting elevated levels of suspended solids (113.6 mg/L), biochemical oxygen demand (BOD, 311.2 mg/L), and chemical oxygen demand (COD, 426.7 mg/L). Real-time PCR detected Giardia spp. DNA in 75% (76/102) of the samples and Cryptosporidium spp. DNA in 94% (96/102) of samples. The metagenomic read-based profiling identified Aeromonas media as a prevalent pathogen and Polaromonas naphthalenivorans as a potential biodegradative agent. The metagenomic assembly produced 270 high-quality genomes, revealing 16 bacterial species (e. g., Acinetobacter johnsonii and Megamonas funiformis) that provided insights into fecal contaminants and native aquatic microbes. Functional profiling further revealed a high prevalence of antimicrobial resistance markers, particularly for tetracyclines, aminoglycosides, and macrolides, with the highest abundance found in wastewater samples. Additionally, virulence factors were notably present in Zoogloea ramigera. The findings underscore the value of metagenomic profiling as a comprehensive tool for water quality monitoring, facilitating the detection of pathogens, beneficial species, and molecular markers indicative of potential health risks. This approach supports continuous monitoring efforts, offering actionable data for water management strategies to safeguard public health and maintain ecological integrity.