BACKGROUNDA fatal case of autochthonous Babesia microti infection was reported in Australia in 2012. This has implications for Australian public health and, given that babesiosis is transfusion transmissible, has possible implications for Australian blood transfusion recipients. We investigated the seroprevalence of antibodies to B. microti in Australian blood donors and in patients with clinically suspected babesiosis.STUDY DESIGN AND METHODSPlasma samples (n = 7,000) from donors donating in at‐risk areas and clinical specimens from patients with clinically suspected babesiosis (n = 29) were tested for B. microti IgG by immunofluorescence assay (IFA). IFA initially reactive samples were tested for B. microti IgG and IgM by immunoblot and B. microti DNA by polymerase chain reaction.RESULTSAlthough five donors were initially reactive for B. microti IgG by IFA, none was confirmed for B. microti IgG (zero estimate; 95% confidence interval, 0%–0.05%) and all were negative for B. microti DNA. None of the patient samples had B. microti IgG, IgM, or DNA.CONCLUSIONSThis study does not provide evidence for widespread exposure to B. microti in Australian blood donors at local theoretical risk, nor does it provide evidence of B. microti infection in Australian patients with clinically suspected babesiosis. Given that confirmed evidence of previous exposure to B. microti was not seen, these data suggest that transmission of this pathogen is currently uncommon in Australia and unlikely to pose a risk to transfusion safety at present.