Aim To investigate the Coxiella burnetii DNA content in environmental samples that may contribute to the transmission of C.burnetii. Methods and Results During a large Q fever outbreak in the Netherlands, surface swabs and aerosol samples were collected inside stables and around six Q fever-affected ruminant farms, which are located in municipalities varying in Q fever incidence. After the outbreak in 2010, aerosol samples were collected in the same geographical areas. The use of an optimized multiplex qPCR for the detection of C.burnetii DNA revealed that all samples obtained inside stables were positive. In addition, the C.burnetii DNA content in aerosol samples collected in stables is significantly higher than in aerosol samples collected around the farms. Finally, the C.burnetii DNA content in aerosol samples collected in the same geographical locations was lower in 2010 in comparison with 2009. Conclusions The reduction in C.burnetii DNA content in aerosol samples between 2009 and 2010 is in agreement with the reduction in Q fever incidence in the same geographical areas. Significance and Impact of the Study The presence of C.burnetii DNA in environmental samples collected on and around ruminant farms supports the hypothesis that C.burnetii can be disseminated from ruminant farms to the surrounding areas.