Background Equine influenza virus (EIV) is considered enzootic in Europe (except Iceland), Asia, North Africa, and North and South America. When EIV outbreaks occur they may severely impact the equine and tourist industries. Australia faced its first EIV outbreak beginning in August of 2007. The outbreak was concentrated in New South Wales and Queensland, with more than 1400 confirmed EIV infections in horses during the first month. Rapid response from the equine industry and the federal government was successful and Australia was declared free from EIV by the end of 2007. Objectives This cross-sectional study was designed to examine associations between exposure to EIV-infected horses and evidence of EIV infection in humans. Study design Employing informed consent, between October 2007 and April 2008, 100 subjects (89 with horse exposures and 11 non-exposed) were enrolled during equine events and at the University of the Sunshine Coast. All subjects provided a blood sample and were asked to complete an online questionnaire including health history, animal exposure and demographic information. Sera samples were tested for the presence of antibodies against two H3N8 EIV strains using microneutralization, hemagglutination inhibition, and enzyme-linked lectin assays. Results Evidence for H3N8 infection was sparse, with only 9 study participants having any indication of H3N8 infection and the seroreactivity seen was low and easily explained by cross-reactions against human influenza strains or vaccines. Conclusions These data provide little evidence to support the premise that EIV infections occurred among humans exposed to EIV-infected horses during the 2007 Australian epizootic.