This study aimed to determine the distribution and relative abundance of freshwater fishes in the Tsitsikamma National Park. Fish assemblages in six river systems were sampled in 2001, with a total of 323 fish from eight species recorded. Indigenous fish collected included four freshwater species (Pseudobarbus afer, Pseudobarbus tenuis, Sandelia capensis, Anguilla mossambica), three estuarine species (Monodactylus falciformis, Caffrogobius gilchristi, Myxus capensis), and one alien (Micropterus salmoides). One additional indigenous species (Galaxias zebratus) and two aliens (Salmo trutta, Oncorhynchus mykiss) could potentially occur within the park. The topography and locality of the park presents a unique opportunity to meaningfully conserve the endangered P. tenuis as well as other fish characteristic of the eastern reaches of the Cape Floristic Region. Management action is required to minimise opportunities for further establishment and spread of alien fish species and to conserve indigenous fish assemblages within the park.