The aim of this study was to evaluate the seroprevalence of Coxiella burnetii in different red deer populations and to investigate role of red deer densities, livestock, and habitat on seroprevalence. The serosurvey revealed 5 positive cases out of 137 sera (3.64%) that occurred in two of the three study areas. This study documents the first cases of Coxiella burnetii in red deer in the southwest Iberian peninsula. A relationship between deer density and Coxiella seroprevalence was not found. Results revealed that indirect transmission through ticks between livestock and red deer might be associated with higher prevalence. The timing of shelter area usage may influence the contact between ticks and red deer by favoring transmission. Coxiella burnetii in red deer may be associated with infertility or early abortions with reabsorption. Further research is needed to evaluate its epidemiology and effect on the disease dynamics of red deer in the southwest Iberian peninsula.