ABSTRACT In Australia, Toxoplasma gondii is an introduced parasite with a wide host range. House cats (Felis catus) are one of its definitive hosts. Little is known about T. gondii infection rates in Australian wildlife. Since cat abundance varies with landscape composition, we hypothesised that T. gondii infection would be more prevalent in urban and agricultural landscapes than intact bushland and sought to use samples from the Southern Boobook (Ninox boobook) as an indicator of ecosystem-wide T. gondii contamination. We used modified agglutination tests to determine T. gondii seropositivity in serum and meat juice samples taken from boobooks. Moderately low levels of seropositivity were detected and non-significant landscape-related patterns of seroprevalence were observed. We also examined correlations and interactions with owl age, season, injury status and exposure to an anticoagulant rodenticide. Only season showed significant correlations with observed seropositivity. To the best of our knowledge, this is the first published detection of T. gondii seropositivity in a wild predatory bird in Australia.