An emerging field of research is starting to examine the association of infectious pathogens with type 2 diabetes mellitus (T2DM). An understudied parasite of interest is Toxoplasma gondii. Globally, very few studies have been conducted to investigate this association. Additionally, very little data exists on the prevalence of T. gondii in the general Australian population. Our group sought to determine the prevalence, association, and risk factors between T. gondii infection and T2DM from a representative Australian human population. Through a cross-sectional, age-A nd gender-matched case-control study, 150 subjects with T2DM together with 150 control subjects from the Busselton Health Study cohort were investigated. Sera samples were tested for the presence of anti-T. gondii IgG and IgM antibodies using enzyme-linked immunosorbent assays. Survey-derived data were also analyzed to evaluate associated risk factors. The IgG seroprevalence was found to be 62% and 66% for the T2DM and control groups, respectively (OR:0.84; p=0.471). IgM antibodies were detected in 5% of the T2DM patients and in 10% of the controls (OR=0.51; p=0.135). There were no significant differences between male and female IgG seroprevalence rates for both groups (OR:0.88, 0.80; p=0.723). The IgG seropositivity rate increased significantly in T2DM patients aged 45-84 years in comparison to those aged 18-44 years (p<0.05), but this was not observed in the control subjects. No risk factors were associated with T. gondii seropositivity in both groups. The first Australian study of its kind found T. gondii infection in Western Australia to be highly prevalent. The results also showed that there is no serological evidence of an association between T. gondii infection and T2DM in the studied subjects. Australian health authorities should focus on raising awareness of toxoplasma infection and target T. gondii transmission control. Further studies are needed to clarify the role of T. gondii in T2DM.