DNA barcodes are instrumental for identifying species from tissues and environmental samples. Metabarcoding-based assessments of biodiversity depend on taxonomically accurate barcode sequence libraries that can be queried to determine which species are present in a sample. The mitochondrial 12S ribosomal RNA gene has been shown to function as a robust barcode for differentiating taxa and identifying species using environmental DNA (eDNA), but complete 12S reference barcodes remain scarce in public databases. We assembled a library of 12S rDNA reference barcodes for 152 species of Neotropical electric fishes from the order Gymnotiformes (Teleostei, Ostariophysi). These fishes are an abundant and diverse component of Neotropical freshwater ecosystems, but are often difficult to collect through conventional sampling approaches because of their nocturnal activity and association with dense substrates or deep-water habitats. This makes eDNA metabarcoding an especially suitable approach for their detection. We compiled a dataset comprising all gymnotiform families, with most sequences covering almost the entire 12S gene. Across the gene and within two widely used 12S eDNA metabarcoding loci (MiFish and teleo), our sequences show sufficient interspecific divergence to discriminate the majority of knifefish species included in our analyses. This dataset, therefore, provides a valuable new set of resources for differentiating amongst gymnotiform species and identifying them in environmental DNA samples.