Ecosystems within cities can contribute significantly to preserving local biodiversity in the face of urban sprawl, but they face significant threats from anthropogenetic activities and the introduction of alien invasive species (AIS). A comprehensive management plan is required to adequately preserve the biodiversity harboured by urban ecosystems. However, ecological information needed to establish, implement and monitor such plans is often incomplete. In this study, we assessed the application of eDNA metabarcoding in surveying plant biodiversity in an aquatic habitat by collecting water samples at five sites in an urban dam in the City of Johannesburg. Out of 178 reconstructed OTUs, only 43 could be assigned to species level (including four AIS). The remaining OTUs could not be identified below the genus level, indicating that no DNA barcodes have yet been generated for the putative species in question. The spatial distribution of plant diversity across the surveyed dam was heterogeneous. Although this study provides a good overview of plant community structure, it also revealed a gap in the taxonomic coverage of South African plants on public DNA databases. This emphasises the need for increased national barcoding efforts to expand current reference sequence databases.