Hybridisation and introgression are natural phenomena that may lead to the transfer of adaptive alleles from one species to another and increased species diversity. At the same time, hybridisation and subsequent introgression threaten many species world-wide through the loss of genetic and species diversity. In Australia, introgressive hybridisation between native and alien species has not typically been considered a significant threat to native biodiversity because of the taxonomic distance between native and alien biota. However, many native fish have been introduced outside their natural range. Recently, four taxa in the genus Melanotaenia have been nationally listed as threatened due to introgressive hybridisation with introduced Melanotaenia splendida. We examined pre- and post-zygotic barriers to hybridisation between M. splendida and one of these threatened taxa—Running River rainbowfish (RRR)—to assess the potential for hybridisation to occur. We used dichotomous mate choice experiments to examine pre-zygotic barriers and mating experiments to examine post-zygotic barriers. Size was not a significant predictor of the proportion of time subjects spent with a potential mate, nor was there any significant difference in the amount of time subjects spent with potential mates of their own or the opposite species. Eggs from hybrid pairings with female RRR had a slightly higher hatching rate than those from hybrid pairings with female M. splendida, but neither were significantly different from intraspecies crosses. We could not identify any definite barriers to hybridisation, demonstrating that the introduction of “native” fish species outside their natural range poses a higher risk of hybridisation than previously thought. We call for better education around the consequences of moving “native” fish and the development of rapid response plans to deal with recently established alien populations of Australian fish species in order to prevent future extinctions due to introgressive hybridisation.