As freshwater sources become increasingly polluted and depleted, the delicate balance of aquatic environments is disrupted, leading to cascading effects throughout entire ecosystems. This disruption manifests in various ways, including changes in water chemistry, temperature fluctuations, and the introduction of contaminants, all of which contribute to alterations in microbial communities. We applied eDNA metabarcoding to characterize microbial communities along an anthropogenic pollution gradient in the Magdalena River, a tropical river in Mexico City. Sampling was conducted at four sites representing different levels of human influence. Results revealed differences in both bacterial and microeukaryotic community compositions between sites. In areas with low to moderate disturbance, bacterial genera associated with nitrogen cycling and plant-microbe interactions (e.g., Rhizobacter, Rhodoferax, and Flavobacterium) were predominant, whereas in more heavily impacted sites, genera linked to enteric, nosocomial, or fecal sources (e.g., Arcobacter, Acinetobacter, and Aeromonas) dominated. Peri-urban sites exhibited higher alpha diversity at the phylum level for bacteria, and microeukaryotic communities; two phyla account for over 75% of the relative abundance throughout the year (Ciliophora & Chlorophyta). Statistical analysis showed that water quality influences microbial composition in the sites. These findings demonstrate that urban influence alter microbial community composition, showing similar patterns to other studies. Our study, however, also discovered certain taxa that had not been previously recorded in tropical urban rivers, thereby broadening the existing knowledge, which has primarily been based on temperate systems. This research offers one of the initial thorough evaluations of microbial communities in urban rivers in Mexico and highlights the potential of eDNA metabarcoding as a valuable tool for environmental monitoring.