The increase in antibiotic resistant bacteria has raised global concern regarding the future effectiveness of antibiotics. Human activities that influence microbial communities and environmental resistomes can generate additional risks to human health. In this work, we characterized aquatic microbial communities and their resistomes in samples collected at three sites along the Bogotá River and from wastewaters at three city hospitals, and investigated community profiles and antibiotic resistance genes (ARGs) as a function of anthropogenic contamination. The presence of antibiotics and other commonly used drugs increased in locations highly impacted by human activities, while the diverse microbial communities varied among sites and sampling times, separating upstream river samples from more contaminated hospital and river samples. Clinically relevant antibiotic resistant pathogens and ARGs were more abundant in contaminated water samples. Tracking of resistant determinants to upstream river waters and city sources suggested that human activities foster the spread of ARGs, some of which were co-localized with mobile genetic elements in assembled metagenomic contigs. Human contamination of this water ecosystem changed both community structure and environmental resistomes that can pose a risk to human health.