Analysis of environmental DNA (eDNA) enables detection of specific species from water and soil samples. Typically, these analyses are performed by amplifying a short DNA sequence using species-specific primers. Alternatively, primers amplifying many species within a taxonomic group are used and amplicons are subsequently sequenced. Here, we describe a method to quickly characterize the biodiversity of a given environment by amplification of eDNA using a combination of primer pairs targeting a wide range of taxa and species identification by high-throughput sequencing. We tested this approach by analyzing 91 water samples of 40mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA extracted from each water sample using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and simultaneously sequenced all PCR products by high-throughput sequencing. Despite the small sample volumes analyzed, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animal species, we identified DNA from many terrestrial species that live near the banks of the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from a small sample of water using wide-range PCR amplification combined with massively parallel sequencing can provide a broad perspective on the biological diversity of a given environment.