Toxoplasma gondii is a cosmopolitan protozoon parasite, and the causative agent of toxoplasmosis, one of the most prevalent zoonotic parasitic diseases. Cats, as definitive hosts, are spreaders of the parasite via their faeces, but this occurs only for a very short period in their life. Seropositivity in cats, although not associated with current shedding of the parasite, is indicative of the infection in a cat population and can be used to assess the infection risk for definitive and intermediate hosts in that area. In order to assess the prevalence of infection in cats living in Cyprus, 155 cats, originating from all districts of the country, were examined for the presence of T. gondii antibodies. Additionally, parameters such as age, sex, health status, lifestyle and concomitant infections were statistically assessed as potential risk factors for T. gondii seropositivity. Specific anti-T. gondii antibodies were detected in 50 (32.3%) cats, while the presence of feline immunodeficiency virus antibodies and a history of never having been vaccinated were statistically associated with T. gondii seropositivity on multivariate logistic regression model. This is the first report of T. gondii seroprevalence in cats in Cyprus and indicates that raised public awareness should be considered to prevent infection of animals and humans.