The epidemiology of Q fever, caused by Coxiella burnetii, varies significantly worldwide. This study aimed to document the prevalence of Coxiella burnetii in Greece by measuring specific IgG antibody levels in serum samples from the general population and high-risk groups, including farmers, veterinarians, and laboratory workers. A multiregional, stratified sampling design was employed, with 1,345 participants from Thessaly and Central Macedonia. Serum samples were tested for Coxiella burnetii IgG antibodies, and multivariate analysis was conducted to identify factors associated with seroprevalence. Overall, 8.1% of participants tested positive for Coxiella burnetii antibodies, with the highest seroprevalence in Larissa (22.2%) and Karditsa (16.1%). High-risk occupational groups, particularly those with direct animal contact, showed a higher seroprevalence (13.6%). Multivariate analysis identified significant associations between seroprevalence and factors such as geographic region, occupation, and gender. The study reveals regional and occupational disparities in Q fever seroprevalence in Greece, particularly in rural areas. These findings underscore the need for targeted public health measures, including heightened surveillance and preventive interventions for high-risk groups.