Due to their frequent contact with animals, veterinarians may be at preferential risk of Coxiella burnetii exposure due to occupational contact with livestock. This study assesses the seroprevalence and risk factors associated with C. burnetii seropositivity in Portuguese veterinarians. A cross-sectional study compared IgG anti-C. burnetii in veterinarians’ sera to a demographically matched control group. Univariate and multivariate logistic regression analyses evaluated associations between the demographic, occupational, and biosecurity factors and seropositivity. Seroprevalence among veterinarians was 33.7%, significantly higher (p = 0.0023) than in the controls (17.39%). Univariate analysis identified higher seropositivity in the northern region (p = 0.03), though this association was not significant after adjustment (p = 0.07). Protective measures, including isolating aborting animals from the rest of the herd (adjusted OR [aOR]: 0.35, p = 0.03) and wearing gloves during sample collection (OR: 0.28, p = 0.009), were significantly associated with lower infection risk. Veterinarians face increased C. burnetii exposure, but specific biosecurity practices reduce risk. Strengthening preventive measures, including personal protective equipment (PPE) use and biosecurity training, is essential to mitigate occupational and public health risks. Further research should explore vaccination strategies and molecular epidemiology to improve risk reduction efforts.