The small, introduced poeciliid Gambusia holbrooki is widely distributed in Australia, and is widespread and generally abundant in lacustrine environments near Perth, Western Australia. Laboratory feeding trials and field investigations were conducted to evaluate the impact of this species on native frogs in southwestern Australia. The eggs of Crinia georgiana, C. insignifera, Litoria adelaidensis, and L. moorei were not consumed by G. holbrooki. In contrast, Crinia georgiana, C. insignifera, C. glauerti, and Litoria moorei hatchlings (Gosner stage 20–23) were consumed by G. holbrooki. Alternative aquatic invertebrate prey (mosquito larvae and Daphnia) were preferred over hatchlings. Gambusia holbrooki were recorded at 20 of 25 wetlands surveyed, and frogs occurred at all 25 sites. From one to six frog species occurred at wetland sites where fish were present. Frog species richness, and the presence of individual species, did not differ between sites with and without G. holbrooki. However, frog species richness was positively correlated with wetland condition (highly ranked sites were relatively undisturbed). In contrast to the situation in eastern Australia, populations of anuran species in southwestern Australia do not appear to be strongly affected by this small invasive fish. This difference is likely related to climatic dissimilarities that affect the population biology of fish and frogs in these regions. Additional factors that may influence the level of impact of G. holbrooki include egg deposition site, timing of anuran breeding, availability of alternative prey, wetland condition, and seasonal variation in fish size and abundance.