Lyme borreliosis (or Lyme Disease) is an emerging threat to human health in the Northern Hemisphere caused by tick-borne bacteria from the Borrelia burgdorferi sensu lato (Bbsl) complex. Seabirds are important reservoir hosts of some members of the Bbsl complex in the Northern Hemisphere, and some evidence suggests this may be true of penguins in the Southern Hemisphere. While the Bbsl complex has not been detected in Australia, a novel Borrelia species ('Candidatus Borrelia tachyglossi') was recently sequenced from native ticks (Ixodes holocyclus and Bothriocroton concolor) parasitising echidnas (Tachyglossus aculeatus), suggesting unidentified borreliae may be circulating amongst native wildlife and their ticks. In the present study, we investigated whether ticks parasitising little penguins (Eudyptula novaehollandiae) harbour native or introduced Borrelia bacteria. We chose this penguin species because it is heavily exploited by ticks during the breeding season, lives in close proximity to other potential reservoir hosts (including native wildlife and migratory seabirds), and is known to be infected with other tick-borne pathogens (Babesia). We screened over 230 penguin ticks (Ixodes spp.) from colonies in south-eastern Australia, and found no evidence of Borrelia DNA. The apparent absence or rarity of the bacterium in south-eastern Australia has important implications for identifying potential tick-borne pathogens in an understudied region.