Ixodes holocyclus (Acarina: Ixodidae) and Ixodes cornuatus (Acarina: Ixodidae) are two tick species found in the more densely populated areas of Australia and are known to be the cause of the neurotoxic disease tick paralysis in humans and mammals. Borreliosis otherwise known as Lyme disease is an emerging infectious disease in humans in Australia. Borrelia burgdorferi sensu stricto (Spirochaetales: Spirochaetaceae) and sensu lato are closely related spirochetal species that are the causative agents of Lyme disease in humans. Clinical transmission of this tick-borne disease can be identified in several but not all cases by a characteristic rash known as erythema migrans. However, there has been no study of the tick vectors of this infection in Australia. We used morphological and molecular techniques to identify unequivocally the ticks on the patients of this study to be I. holocyclus and then show the presence of B. burgdorferi sensu stricto infection in erythema migrans biopsies. I. holocyclus has not previously been associated with erythema migrans or Lyme disease. Two patients presented to the lead author's medical practice with erythema migrans in mid and late 2012. The morphology and cytochrome oxidase 1 and ITS2 genes of the two ticks were studied. The skin at the attachment site was sampled by central biopsy for both real time and endpoint Borrelia polymerase chain reaction (PCR) analysis and subsequent sequencing. Morphologically, the two ticks were either I. holocyclus or I. cornuatus. Molecular studies and nucleotide sequencing revealed that both ticks were I. holocyclus. Real time and endpoint PCR on the central tissue biopsy samples returned positive results for B. burgdorferi DNA. Our results are evidence for transmission of B. burgdorferi sensu stricto species to humans by the tick I. holocyclus in Australia. I. holocyclus is commonly associated with human tick bites on virtually the entire eastern coastline of Australia.