The Paraguayan Chaco, situated in western Paraguay, is characterized by an arid ecosystem and a limited human population. The Isla Po'i Lagoon, situated in the Department of Boquerón, is a key ecological feature that serves as one of the few perennial water sources in times of drought and provides a vital habitat for a diverse range of species. Microorganisms are integral components of aquatic ecosystems, and their abundance varies based on ecological niches. Traditional culture-based methods are limited by their ability to explore microbial diversity. High-throughput sequencing has significantly transformed environmental microbiology, facilitating direct study of microbial communities in their natural habitats. This study employed long-read sequencing using MinION technology to investigate the microbial diversity in the Isla Po'i Lagoon. To determine taxonomic composition, analyses were conducted based on sequencing reads. We observed an average diverse community composition comprising bacteria (86.2%), eukaryota (11.6%), viruses (2%), and archaea (0.2%). The dominant phylum identified across all classifiers was Proteobacteria (44.3-94.0%), which underscores its importance in various aquatic environments. Nanopore sequencing provided a detailed characterization of the microbial community, offering insights into the diversity of the Isla Po'i Lagoon.