Interoceanic canals can facilitate biological invasions as they connect the world's oceans and remove dispersal barriers between bioregions. As a consequence, multiple opportunities for biotic exchange arise and the resulting establishment of migrant species often causes adverse ecological and economic impacts. The Panama Canal is a key region for biotic exchange as it connects the Pacific and Atlantic Oceans in Central America. In this study, we used two complementary methods (environmental DNA (eDNA) metabarcoding and gillnetting) to survey fish communities in this unique waterway. Using COI (cytochrome oxidase subunit I) metabarcoding, we detected a total of 142 fish species, including evidence for the presence of sixteen Atlantic and eight Pacific marine fish in different freshwater sections of the Canal. Of these, nine are potentially new records. Molecular data did not capture all species caught with gillnets, but generally provided a more complete image of the known fish fauna as more small-bodied fish species were detected. Diversity indices based on eDNA surveys revealed significant differences across different sections of the Canal reflecting in part the prevailing environmental conditions. The observed increase in the presence of marine fish species in the Canal indicates a growing potential for interoceanic fish invasions. The potential ecological and evolutionary consequences of this increase in marine fishes are not only restricted to the fish fauna in the Canal as they could also impact adjacent ecosystems in the Pacific and Atlantic Oceans.