Background: Large imaging systems in hybrid operating rooms are used increasingly. However, the effect of these ceiling-mounted imaging systems on air quality during surgical procedures has not been studied to date. Aim: To evaluate the level of colony-forming units (cfu)/m3 near the surgical wound and near the instrument table during surgery. Methods: Measurements were performed in four hybrid operating rooms at four different hospitals. During surgical procedures, at least three samples were taken with active slit air samplers. At the end of the day's surgical schedule, simulations involving movement of the ceiling-mounted system were also performed. The threshold value for the average cfu level during a surgical procedure was set at ≤10 cfu/m3, and for individual samples was set at ≤30 cfu/m3. Results: The median value near the surgical wound was 1 cfu/m3 and at the instrument table was 2 cfu/m3. However, in one hybrid operating room (two procedures out of 16) where the instrument table was not positioned directly under the unidirectional flow (UDF) system, the threshold value for the average cfu level at the instrument table was exceeded. For one of these procedures, the maximum value for an individual sample was also exceeded. Conclusions: A ceiling-mounted imaging system in combination with a UDF system can result in cfu levels near the surgical wound and at the instrument table that are well below the threshold value of 10 cfu/m3 during surgery. If the instrument table is not positioned directly under the UDF system, the cfu level is higher.