AbstractCharacterizing biological communities and knowledge on the distribution of biodiversity allows the assessment of ecological quality. This provides valuable information for conservation biology and monitoring purposes. While obtaining such data has been challenging in the past, environmental DNA (eDNA) sampling represents a promising tool to describe biodiversity on a broad taxonomic scale. In this study, we provide the first broad‐scale biodiversity assessment for ten Neotropical water bodies in Nicaragua (a major river, two great lakes, and seven relatively young and small crater lakes) using eDNA sampling to determine how abiotic factors structure the distribution of prokaryotic and eukaryotic biodiversity across these environments. Further, we explored to what extent levels of biodiversity are associated across different taxonomic groups and environments. We found that prokaryotic and eukaryotic α‐diversity was consistently higher in the great lakes (i.e., Lakes Nicaragua and Managua) as well as in Río San Juan compared with the young and small crater lakes. Differences of prokaryotic and eukaryotic communities (β‐diversity) were significantly correlated, indicating that biological communities are similarly structured across environments. Accordingly, differences in salinity were correlated with prokaryotic and eukaryotic communities, whereas differences in dissolved oxygen were only correlated with prokaryotic communities (β‐diversity). Yet, salinity and dissolved oxygen only affected prokaryotic α‐diversity, suggesting different effects of these two abiotic factors on biodiversity within aquatic environments. Moreover, α‐diversity of different phyla was positively correlated, although more strongly in prokaryotes, showing that biodiversity patterns are congruent across a broad range of lineages, particularly in prokaryotes.