Freshwater is an essential resource on our planet; however, human activities have significantly impacted it. This study aimed to assess the microbiome of Pasto River's wastewater and surface waters in the southwestern Colombian Andean highlands. Traditional physicochemical and microbiological analyses, alongside Giardia spp. and Cryptosporidium spp parasites were detected by real time PCR. Illumina technology was used for metagenomic analyses. allowing to explore both taxonomic and functional profiling using two approaches. The first approach was from reads reveling the most abundant families, along with species exhibiting potential pathogenic characteristics, like Aeromonas media, and those with potential beneficial roles, such as Polaromonas naphthalenivorans. It also uncovered molecular markers of health significance, including common antimicrobial resistances markers linked to tetracyclines and aminoglycosides, as well as virulence factors. The second approach involved genome assembly using metagenomic data, leading to 270 high-quality assemblies and the identification of 16 bacterial species, encompassing both fecal-derived and water-body-native species, including insights into their functional relationships. This study provides a current state of the Pasto River, encompassing both surface and untreated wastewater, and offers a valuable tool for assessing potential risks associated with water reuse and the implications of the direct wastewater discharge into surface waters bodies.
