This study is focused on the aquatic environments of the Sian Ka’an reserve, a World Heritage Site. We applied recently developed protocols for the rapid assessment of most animal taxa inhabiting any freshwater system using light traps and DNA barcodes, represented by the mitochondrial gene Cytochrome Oxidase I (COI). We DNA barcoded 1037 specimens comprising mites, crustaceans, insects, and fish larvae from 13 aquatic environments close or inside the reserve, with a success rate of 99.8%. In total, 167 barcode index numbers (BINs) were detected. From them, we identified 43 species. All others remain as a BIN. Besides, we applied the non-invasive method of environmental DNA (eDNA) to analyze the adult fish communities and identified the sequences obtained with the Barcode of Life Database (BOLD). All round, we found 25 fish species and other terrestrial vertebrates from this region. No alien species was found. After comparing the BINs from all systems, we found that each water body was unique with respect to the communities observed. The reference library presented here represents the first step for future programs to detect any change in these ecosystems, including invasive species, and to improve the knowledge of freshwater zooplankton, enhancing the task of compiling the species barcodes not yet stored in databases (such as BOLD or GenBank).